Catabolic nanocompartments

Abstract

A method of breaking down at least one component of a substrate can include providing a catabolic nanocompartment that includes a liquid coacervate phase, at least one catalyst within the coacervate phase, and an aqueous phase surrounding the coacervate phase. The method can include contacting the catabolic nanocompartment with the substrate including the at least one component such that the catalyst degrades the at least one component and forms degradation products of the at least one component.

Description

CATABOLIC NANOCOMPARTMENTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 719,484 filed 11 / 12 / 2024, the disclosure of which is incorporated herein in its entirety by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number 2421209 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND

[0003] Extreme weather and decades of fire suppression policies have created conditions that make much of the western United States highly susceptible to devastating wildfires. Recent years have seen longer fire seasons resulting in costly losses of property7, livelihoods, habitat, biodiversity, and watershed health. Current forest management practices across the Western US focus on strategic thinning followed by controlled burning of the resulting slash (small trees, branches, and debris). However, these controlled bum activities have sometimes resulted in the ignition of catastrophic mega-wildfires, causing significant loss of life and property along with environmental and economic degradation.

[0004] The current practice of burning thinned slash represents an unprofitable waste of potentially valuable biomass. Coniferous forests contain significant stores of potential chemical feedstocks that could, in principle, be readily converted to valuable products through fermentation and synthetic biological methods. However, these feedstocks are present in wood in a refractory complex matrix with lignin, which has historically been difficult to utilize through existing naturally derived, green industrial processes. While thermochemical methods have been developed for lignin degradation to unlock cellulose-based feedstocks from wood, these approaches often pose environmental and safety risks that make them unsustainable. The primaryimpediment to biochemical access to these precursors is their encapsulation and close association with lignin — a chemically recalcitrant, heterogeneous macromolecule with little regularity and historically low commercial value. Existing biochemical approaches using oxidases (specifically laccases) and peroxidases found in fungi and bacteria have shown some promise for breaking down lignin, as these organisms are nature’s primary forest product recyclers. However, a significant limitation of traditional enzymatic methods for lignin degradation is the tendency for oxidized lignin subunits to quickly recombine (repolymerize) after they form, which hampers breakdown efficiency.

[0005] There is therefore a significant need for new approaches to dispose of thinned slash while simultaneously addressing the growing need to replace petroleum-based feedstocks to reduce greenhouse gas emissions that imperil forest health through extreme weather. Development of effective green methodologies for overcoming the “lignin barrier’’ could enable many promising yet still challenging processes for biomass utilization, including the production of biofuels and commodity / specialty chemicals through recently developed green biosynthetic methodologies.SUMMARY OF THE INVENTION

[0006] Various aspects of the present disclosure provide a method of breaking down at least one component of a substrate. The method includes providing a catabolic nanocompartment including a liquid coacervate phase, at least one catalyst within the coacervate phase, and an aqueous phase surrounding the coacervate phase. The method includes contacting the catabolic nanocompartment and the substrate together such that the catalyst breaks down at the least one component of the substrate and forms degradation products of the at least one component.

[0007] Various aspects of the present disclosure provide a method of degrading lignin, such as on its own or in woody biomass. The method includes providing a catabolic nanocompartment including a liquid coacervate phase, at least one laccase enzy me within the coacervate phase, and an aqueous phase surrounding the coacervate phase. The method includes contacting the catabolic nanocompartment with a lignin or with a woody biomass particle includinglignin such that the laccase enzy me degrades the lignin and forms lignin degradation products.

[0008] Various aspects of the present disclosure provide a catabolic nanocompartment composition including a liquid coacervate phase including at least one catalyst. The catabolic nanocompartment composition also includes an aqueous phase surrounding the coacervate phase. The composition degrades at least one component of a substrate to form degradation products of the at least one component when the at least one component is contacted with the liquid coacervate phase.

[0009] Various aspects of the present disclosure provide a catabolic nanocompartment composition including a liquid coacervate phase including at least one laccase enzy me. The composition also includes an aqueous phase surrounding the coacervate phase. The composition degrades lignin to form lignin degradation products when the lignin is contacted with the liquid coacervate phase.

[0010] Various aspects of the present disclosure provide a method of making a catabolic nanocompartment composition. The method includes providing an aqueous solution including coacervate-forming components. The method also includes adding at least one catalyst to form a liquid coacervate phase containing the catalyst, to form the catabolic nanocompartment composition. The catabolic nanocompartment composition degrades at least one component of a substrate to form degradation products of the at least one component when the at least one component is contacted with the liquid coacervate phase.

[0011] Various aspects of the present disclosure provide a method of making a catabolic nanocompartment composition. The method includes providing an aqueous solution including coacervate-forming components. The method also includes adding at least one laccase enzyme to form a liquid coacervate phase containing the laccase enzyme, to form the catabolic nanocompartment composition. The catabolic nanocompartment composition degrades lignin to form lignin degradation products when the lignin is contacted with the liquid coacervate phase.

[0012] Various aspects of the present disclosure provide various advantages across technical, environmental, economic and social dimensions.From a technical perspective, the catabolic nanocompartment technology can enable highly efficient enzymatic degradation of lignin by overcoming several key limitations of existing approaches. The fluid coacervate-based nanoreactors can spontaneously coat lignin-containing substrates through temperature-triggered liquid-liquid phase separation, bringing concentrated oxidase enzymes directly into contact with the lignin surface while preventing the repolymerization of oxidized lignin subunits that typically hampers breakdown efficiency. The coacervate phase can selectively recruit and concentrate both enzyme catalysts and redox mediators into the same microenvironment through favorable partitioning interactions, establishing kinetically favorable spatial arrangements that can minimize diffusional barriers and support sequential electron-transfer reactions. The system can maintain favorable conditions for enzymatic activity while effectively solubilizing and initially retaining lignin degradation products within the condensed phase before controlled release. The coacervate phases can be engineered with features like lignin-binding domains to promote selective adhesion to woody substrates and cellulose binding domains to enhance spreading over wood particles. The platform can accommodate both enzyme “clients” that partition into coacervates through noncovalent interactions and enzyme “hostages” that are covalently linked to coacervate-forming polymers, and can be adaptable to different biomass feedstocks beyond coniferous woody waste including various types of extracted and technical lignins.

[0013] Environmental benefits of various aspects of the present disclosure can be substantial, as the technology can provide a green, environmentally-friendly alternative to thermochemical lignin degradation methods that often involve toxic reagents and byproducts. The coacervate-based approach can function under mild temperature and pressure conditions using naturally -derived or recombinantly -produced components. By enabling sustainable production of wood-derived chemical products and replacement of petroleum-based feedstocks, the invention can help reduce greenhouse gas emissions while providing an alternative to slash pile burning that can trigger catastrophic wildfires. The technology can facilitate access to cellulosic materials for green biosynthetic methodologies while avoiding the implementation of genetically modified organisms in field biorefinery settings.

[0014] Economic advantages of various aspects of the present disclosure can be particularly compelling. The technology can create value from waste biomass that would otherwise be burned, potentially helping to offset the costs of forest thinning, fire prevention, and carbon emissions. The platform can enable production of valuable products like biofuels, bioplastics, textiles, pharmaceuticals, and specialty chemicals from waste material. The modular design can allow for cost-effective implementation, as simpler bacterial laccases and synthetic polymers may prove economically competitive with more complex fungal enzy mes, and certain coacervate-forming fusion proteins can facilitate enzyme purification to reduce production costs. The platform's applications can extend beyond lignin deconstruction to areas like decontamination, recycling, biofuel cell catalysis, and enzymatic therapies. Various aspects of the present invention can align with government incentives for clean energy7and bioeconomy development while supporting the profitable utilization of biomass that currently represents a fire hazard.

[0015] From a social impact perspective, various aspects of the present disclosure can help protect vulnerable forest communities from catastrophic wildfires while creating new economic opportunities in disadvantaged nearforest communities. Various aspects of the present disclosure can support the preservation of traditional forest-dependent cultures and provide safety benefits by reducing risks associated with controlled burning of slash piles that have historically ignited devastating mega-wildfires. The technology7can avoid the dangerous high-temperature, high-pressure conditions and toxic chemicals associated with thermochemical methods while helping to mitigate fire danger to lives, property, watersheds, agricultural productivity, and community cohesion. By enabling profitable processing of thinned biomass, the technology can create economic incentives that support sustainable forest management practices.BRIEF DESCRIPTION OF THE FIGURES

[0016] The drawings illustrate generally, by way7of example, but not by7way of limitation, various embodiments of the present invention.

[0017] FIG. 1 A illustrates a photograph of thinned and piled slash, in accordance with various aspects of the present disclosure.

[0018] FIG. IB illustrates a photograph of thinned and piled slash after inadvertent ignition (white spots correspond to burnt slash piles), in accordance with various aspects of the present disclosure.

[0019] FIG. 1C illustrate a scheme for a green bioprocessing system, in accordance with various aspects of the present disclosure.

[0020] FIG. 2A illustrates a schematic of components of a catabolic nanocompartment (CatnC), in accordance with various aspects of the present disclosure.

[0021] FIG. 2B illustrates a typical lignin degradation reaction achieved by laccase (note that implementation of redox mediators within the CatnC can enable laccase-based cleavage of phenolic carbon-carbon bonds as well), in accordance with various aspects of the present disclosure.

[0022] FIG. 3 illustrates fluorescence microscopy images aqueous microdroplets containing lignin particles and LB2-80 and the temperaturedependent LLPS of AF488-labeled LB2-80 and its spatial association with kraft lignin particles, in accordance with various aspects of the present disclosure.

[0023] FIG. 4 illustrates fluorescence microscopy images showing temperature-dependent LLPS of AF488-labeled LB2-80 and its spatial association with wood particles, which autofluoresce in Cy3, in accordance with various aspects of the present disclosure.

[0024] FIG. 5 illustrates agarose gel electrophoresis showing concentration-dependent retardation of lignin migration by the LB2-80 protein at various concentrations, in accordance with various aspects of the present disclosure.

[0025] FIG. 6 illustrates extinction measurements at 330 nm showing solubilized lignin before phase separation (control) and after partitioning between the coacervate (pellet) and supernatant fractions for El -40 and LB2-80 in sodium acetate and PBS buffers, in accordance with various aspects of the present disclosure.

[0026] FIG. 7 illustrates fluorescence microscopy images showing colocalization of LB2-80 coacervates with T. versicolor laccase (labeled with Cy3) and SLAC1 (labeled with AF488), in accordance with various aspects of the present disclosure.

[0027] FIG. 8 illustrates fluorescence measurements at 530 / 570 nm showing Cy-3 labeled T. versicolor laccase before (control) and after partitioning between the coacervate (pellet) and supernatant fractions for El -40 and LB2-80 in sodium acetate and PBS buffers, in accordance with various aspects of the present disclosure.

[0028] FIG. 9 illustrates extinction measurements at 340 nm showing before (control) and after ABTS partitioning between the coacervate (pellet) and supernatant fractions for LB2-80 in sodium acetate, in accordance with various aspects of the present disclosure.

[0029] FIG. 10A illustrates quantitative analysis of ABTS oxidation with laccase in the presence of LB2-80 as measured by extinction at 420 nm. in accordance with various aspects of the present disclosure.

[0030] FIG. 10B illustrates photographs showing macroscopic appearance of laccase- ABTS reactions with LB2-80 after 10 h incubation above Tt in microcentrifuge tubes, in accordance with various aspects of the present disclosure.

[0031] FIG. 10C illustrates photographs showing macroscopic appearance of laccase- ABTS reactions with LB2-80 after 10 h incubation above Tt in microtiter wells, in accordance with various aspects of the present disclosure.

[0032] FIG. 1 1 A illustrates fluorescence microscopy images illustrating the interactions between lignin, Cy3-labeled T. versicolor laccase, and AF488-labeled LB2-80 in microdroplets, in accordance with various aspects of the present disclosure.

[0033] FIG. 1 IB illustrates fluorescence microscopy images illustrating the interactions between lignin, Cy3-labeled T. versicolor laccase, and AF488-labeled LB2-80 in bulk solution, in accordance with various aspects of the present disclosure.

[0034] FIG. 12A illustrates fluorescence microscopy images illustrating temperature-dependent LLPS of AF488-labeled SLAC1.LB2-80 and its spatial association with kraft lignin particles, which autofluoresce in the Cy3 channel, in accordance with various aspects of the present disclosure.

[0035] FIG. 12B illustrates fluorescence microscopy images illustrating temperature-dependent LLPS of AF488-labeled SLAC1 control and a lack ofspatial association with kraft lignin particles, which autofluoresce in the Cy3 channel, in accordance with various aspects of the present disclosure.

[0036] FIG. 13 illustrates fluorescence microscopy images showing the formation of CatnCs with Cy3-fluorescent lignin, AF488-labeled LB2-80, T. versicolor laccase, and ABTS, in accordance with various aspects of the present disclosure.

[0037] FIG. 14 illustrates fluorescence microscopy images of aqueous microdroplets in oil indicate time-dependent bleaching of lignin (which autofluoresces in the microscopes Cy3 channel) in the presence of laccase, ABTS, and LB2-80 over 24 hours, in accordance with various aspects of the present disclosure.

[0038] FIG. 15 A illustrates fluorescence imaging showing the loss of lignin fluorescence in a CatnC sample containing LB2-80, T. versicolor laccase, and ABTS, in accordance with various aspects of the present disclosure.

[0039] FIG. 15B illustrates fluorescence imaging showing the loss of lignin fluorescence in a CatnC sample containing T. versicolor laccase and ABTS (control), in accordance with various aspects of the present disclosure.

[0040] FIG. 16 illustrates fluorescence microscopy images depicting the appearance of product fluorescence during lignin oxidation within LB2-80-based CatnCs containing Cy3-autofluorescent lignin and AF488-labeled LB2-80 at 10 °C and 37 °C, in accordance with various aspects of the present disclosure.

[0041] FIG. 17A illustrates quantitative analysis of lignin degradation in the presence of LB2-80 and laccase, after 10 h incubation at 26 °C (above the phase separation temperature (Tt)). measured by extinction at 340 nm, in accordance with various aspects of the present disclosure.

[0042] FIG. 17B illustrates photographs showing macroscopic appearance of laccase-lignin reactions in the presence of LB2-80 after 10 h incubation above the phase separation temperature (Tt) in microcentrifuge tubes, in accordance with various aspects of the present disclosure.

[0043] FIG. 17C illustrates photographs showing macroscopic appearance of laccase-lignin samples in the presence of LB2-80 after 10 h incubation above the phase separation temperature (Tt) in microtiter plate wells, in accordance with various aspects of the present disclosure.

[0044] FIG. 17D illustrates photographs showing macroscopic appearance of control, laccase-free samples including lignin and LB2-80 after 10 h incubation above the phase separation temperature (Tt) in microtiter plate wells, in accordance with various aspects of the present disclosure.

[0045] FIG. 18 illustrates extinction measurements at 450 nm illustrating the intensity for a lignin control, bulk solution reaction control, and between the coacervate (pellet) and supernatant fractions for LB2-80, in accordance with various aspects of the present disclosure.DETAILED DESCRIPTION OF THE INVENTION

[0046] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0047] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1 % to about 5%” or “about 0.1 % to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0048] In this document, the terms “a," “an," or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of sectionheadings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section.

[0049] In the methods described herein, the acts can be carried out in a specific order as recited herein. Alternatively, in any aspect(s) disclosed herein, specific acts may be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately or the plain meaning of the claims would require it. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0050] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0051] The term “substantially” as used herein refers to a majority' of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.Method of degrading lignin.

[0052] Various aspects of the present disclosure provide a method of breaking down (e.g., degradation and / or catabolism) at least one component of a substrate. The method can include providing a catabolic nanocompartment including a liquid coacervate phase, at least one catalyst within the coacervate phase, and an aqueous phase surrounding the coacervate phase. The method caninclude contacting the catabolic nanocompartment with the substrate including the at least one component such that the catalyst breaks down the at least one component of the substrate and forms degradation products of the at least one component. Repolymerization of the degradation products of the at least one component can be decreased and / or prevented compared to exposure of the substrate to the at least one catalyst outside of the catabolic nanocompartment. The catabolic nanocompartments can spontaneously coat the substrate and bring the at least one catalyst into close proximity thereto. The catabolic nanocompartments can allow the transport of the degradation produce of the at least one component away from the substrate surface to prevent recombination with the substrate.

[0053] Coacervates are polymer-rich aqueous phases (i.e., polymer dense phases) that form spontaneously and separate from polymer-poor phases (i.e., dilute or supernatant phase). Coacervates can form droplets or can spread over solid or liquid objects and can be described as liquid compartments because they can coalesce or flow readily and can spontaneously coat objects.

[0054] The substrate can be any suitable substrate that includes at least one component that can be degraded in the presence of the at least one catalyst. The substrate can be in a solid form, a liquid form, a dissolved form, or a combination thereof. The substrate can include any organic or inorganic material susceptible to chemical, enzymatic, or biological modification, degradation, or transformation, such as a natural, synthetic, environmental, or biological substance or a mixture thereof. The substrate can be or include an enzy me, a substratum (e.g.. a solid), or a combination thereof. The substrate can include a biomass product, a synthetic material to be decomposed or recycled, an environmental contaminant, a toxic contaminant, a biological molecule, a pathological biological entity (e.g., pathogens or cancer cells), or a combination thereof. The substrate can include solid lignin, solubilized lignin, solid kraft lignin, solubilized kraft lignin, extracted lignin, ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), wood, wood particles, woody biomass, or a combination thereof. The substrate can include woody biomass. In various aspects, the substrate includes wood, wood particles, woody biomass, or a combination thereof, and the at least one component includes lignin, wherein the degradation products of the at least one component include lignin degradationproducts. In various aspects, the method can include milling a substrate precursor into particles to form the substrate that is contacted with the catabolic nanocompartment. In various aspects, the method can include collecting the degradation products of the at least one component.

[0055] The catalyst can be any suitable catalyst that can break dow n (e.g., degrade and / or catabolize) the at least one component of the substrate. The catalyst can include an enzyme, a non-enzymatic catalyst (e.g., a metal-organic compound), or a combination thereof. The catalyst can include an enzyme. The catalyst can include an enzyme including a laccase, a small laccase (SLAC), a peroxidase, or a combination thereof. The catalyst can be free of covalent attachment to polymers in the coacervate phase and can be partitioned into the coacervate phase through favorable noncovalent interactions with the coacervate phase and free of covalent attachment to one or more polymers in the coacervate phase (e.g., “clients”). The catalyst can include a laccase including a fungal laccase (e.g., from Trametes versicolor, Myceliophthora thermophila, Pleurotus ostreatus)', a bacterial laccase, an engineered variants with enhanced thermal or pH stability, or a combination thereof. The catalyst can include a peroxidase, such as a heme-dependent enzy me that uses hydrogen peroxide as an oxidant. The peroxidase can include a lignin peroxidase, a manganese peroxidase, a versatile peroxidase, a dye-decolorizing peroxidase, a horseradish peroxidase, or a combination thereof. The peroxidase can can catalyze oxidative depolymerization, cross-linking, or modification of the at least one component such as lignin under appropriate conditions. The catalyst can include a SLAC including bacterial or recombinant monomeric and dimeric laccases derived from Streptomyces sp. (e.g., S. coelicolor and S. azureus), synthetic SLAC analogs, or a combination thereof. SLACs can exhibit high stability' and activity toward phenolic substrates in both aqueous and biphasic systems. The catalyst can be covalently attached to one or more polymers in the coacervate phase (e.g., “hostages”); for example, the catalyst can include a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP), a small laccase / elastin-like poly peptide (SLAC-ELP), or a combination thereof. The SLAC-LB-ELP can be based on the LB series [(VPGXG)mLB]nwherein X is any suitable amino acid, wherein m is 5 to 20 such as 5, 10, 15, or 20. and wherein n is 1 to 50 such as 4.6, or 8, such as [(VPGVG)ioLB]nelastin-like polypeptides, wherein LB is LB1 =HFPSPIFQRHIH or LB2 = HFPSP, in which the small laccase enzyme SLAC1 is genetically fused to the N-terminus of the LB-ELP sequence. For example. SLAC1.LB1-40 corresponds to SLAC1 fused to [(VPGVG)IOLB]4. These constructs serve as proteins that retain lignin binding activity, thermal responsiveness of the ELP and enzymatic laccase activity7. The SLAC-LB-ELP can be based on the LB series SLACl-[(VPGXG)mLB]nwherein X is any suitable amino acid, wherein m is 5 to 20 such as 5, 10, 15, or 20, and wherein n is 1 to 50 such as 4, 6, or 8, such as SLACl-[(VPGVG)ioLB]n. The SLAC-ELPs can be based on the ELP series [(VPGVG)n] elastin-like polypeptides, wherein n is 1 to 500 such as 40, 80, or 160, in which the small laccase enzyme SLAC1 is genetically fused to the N-terminus of the ELP sequence. For example, SLAC1. El-40 corresponds to SLAC1 fused to (VPGVG)40, and SLAC1.E1-80 to SLAC1 fused to (VPGVG) so. These constructs serve as non-binding control proteins that retain the thermal responsiveness of the ELP domain and enzymatic activity but lack the lignin-binding peptide insert present in the LB-ELP variants. The SLAC-LB-ELP or SLAC-ELP can include SLAC1.LB1-40, SLAC1.LB1-60, SLAC1.LB1-80, SLAC1.LB2-40, SLAC1.LB2-60, SLAC1.LB2-80, SLAC. El-40, SLAC. El-80, or a combination thereof. In various aspects, the catalyst is redox active and can work synergistically with one or more redox mediators in the coacervate phase.

[0056] In various aspects, the coacervate phase includes one or more polymers. The one or more polymers can undergo liquid-liquid phase separation and / or undergo exhibit lower or upper critical solubility temperatures. The one or more polymers can have epitopes that interact (specifically or nonspecifically) with the substrate. Polymer-rich coacervates that include catabolic nanocompartments can favorably influence local dielectric properties so that catabolic redox reactions are favored. The one or more polymers can include one or more water-soluble polymers. The one or more polymers can include a synthetic polymer, a recombinantly synthesized biopolymer, or a combination thereof. The one or more polymers can include a mussel foot protein, a resilin-like peptide, an elastin-like peptide, poly( / V-isopropyl acry lamide), polylysine, polyglutamic acid, poly(2-dimethylamino)ethyl methacrylate, gum Arabic, polyethyleneimine, polystyrene sulfonate, polyethylene glycol), poly(acrylic acid), a lignin-binding elastin-like polypeptide (LB-ELP), a cellulose-bindingelastin-like polypeptide (CB-ELP), a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP), a small laccase / elastin-like polypeptide (SLAC-ELP), or a combination thereof. The one or more polymers can include a mussel foot protein, a resilin-like peptide, an elastin-like peptide, poly(N-isopropyl acry lamide), or a combination thereof. The one or more polymers can include one or more pairs of polymers chosen from polylysine / poly glutamic acid, poly(2-dimethylamino)ethyl methacrylate / gum arabic, polyethyleneimine / polystyrene sulfonate, and poly(ethylene glycol) / poly(acrylic acid). The one or more polymers can include one or more lignin-binding elastinlike polypeptides (LB-ELP), such as any suitable LB-ELP described herein. LB-ELPs can host (e.g., concentrate and / or partition) catalysts therein such as enzymes. The one or more polymers can include an LB-ELP based on the ELP El-n (i.e., (VPGVG)n), wherein n is 1 to 500, with one lignin binding peptide inserted every 10 (VPGVG) repeats; for example, each El-10 unit can have either a 12 (LB1) or 5 (LB2) amino acid lignin-binding peptide fused at its end, wherein LB1 = HFPSPIFQRHIH and LB2 = HFPSP. The one or more polymers can include an LB-ELP based on the ELP (VPGV G)ioLB]n, wherein n is 1 to 50, and wherein LB is LB 1 or LB2. The one or more polymers can include one or more LB-ELPs chosen from LB 1-40. LB 1-60, LB 1-80, LB2-40. LB2-60, LB2-80, or a combination thereof. The one or more polymers can include a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP) or a small laccase / elastin-like polypeptide (SLAC-ELP), such as any suitable SLAC-LB-ELP or SLAC-ELP described herein. The SLAC-LB-ELP or SLAC-ELP can include SLAC1.LB1-40, SLAC1.LB1-60, SLAC1.LB1-80, SLAC1.LB2-40, SLAC1.LB2-60, SLAC1.LB2-80, SLAC.E1-40, SLAC.E1-80, or a combination thereof.

[0057] In various aspects, the coacervate phase can include a redox mediator. The redox mediator can facilitate electron transfer between the catalyst and the substrate, enabling oxidation of high-molecular- weight or sterically inaccessible domains of the substrate. The redox mediator can include 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldehyde (SGA), 1 -hydroxybenzotriazole (HBT), 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), 2,6-dimethoxyphenol (DMP), or a combination thereof.

[0058] The aqueous phase can include a deep eutectic solvent. The deep eutectic solvent can include a hydrogen bond donor and a hydrogen bond acceptor. The hydrogen bond donor can be any suitable hydrogen bond donor, such as ethylene glycol, glycerol, erythritol, xylitol, or propionic acid. The hydrogen bond acceptor can be any suitable hydrogen bond acceptor, such as choline chloride, choline dihydrogen citrate, or a betaine.

[0059] The coacervate phase can include a cellulose binding domain. The coacervate phase can include an oxygen reservoir. The oxygen reservoir can include silicone nanoparticles. The coacervate phase can include a spreading promoter.

[0060] Various aspects of the present disclosure provide a method of degrading lignin in woody biomass. The method can include providing a catabolic nanocompartment including a liquid coacervate phase, at least one laccase enzy me within the coacer ate phase, and an aqueous phase surrounding the coacervate phase. The method can include contacting the catabolic nanocompartment with a woody biomass particle including lignin such that the laccase enzyme degrades the lignin and forms lignin degradation products. Repolymerization of the lignin degradation products can be decreased and / or prevented compared to exposure of the woody biomass particle to the one or more laccase enzymes outside of the catabolic nanocompartment.

[0061] The laccase enzyme can include any suitable laccase enzyme. The laccase enzyme can include a small laccase (SLAC). The laccase enzyme can include a fusion protein. The fusion protein can include a laccase domain and a coacervation tag domain.

[0062] The coacervate phase can include at least one component chosen from polylysine, polyglutamic acid, mussel foot protein, resitin-like peptides, elastin-like peptides, poly(2-dimethylamino)ethyl methacry late, gum arabic, polyethyleneimine, polystyrene sulfonate, poly(ethylene glycol), poly(aciylic acid), and poly(N-isopropyl acrylamide).

[0063] The coacervate phase can include at least one redox mediator. The redox mediator can be any suitable redox mediator. The redox mediator can be chosen from 1 -hydroxybenzotriazole (HBT) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).

[0064] The woody biomass particle can be obtained in any suitable way. The method can include milling woody materials into particles to form the woody biomass particle.

[0065] The method can further include collecting the lignin degradation products.Catabolic nanocompartment composition.

[0066] Various aspects of the present disclosure provide a catabolic nanocompartment composition. The catabolic nanocompartment composition can be any suitable composition that includes the catabolic nanocompartments described for use in the method of the present disclosure. For example, the catabolic nanocompartment composition can include a liquid coacervate phase including at least one catalyst. The catabolic nanocompartment composition can also include an aqueous phase surrounding the coacervate phase. The composition can degrade at least one component of a substrate to form degradation products of the at least one component of the substrate when the at least one component is contacted with the liquid coacervate phase. In various aspects, when the at least one component is contacted with the liquid coacervate phase and the degradation products of the at least one component are formed, repolymerization of the degradation products of the at least one component is decreased and / or prevented compared to exposure of the at least one component to the one or more catalysts outside of the catabolic nanocompartment. The catabolic nanocompartment composition can include the same catabolic nanocompartments as described herein for use in the method of breaking down at least one component of a substrate. The components of the catabolic nanocompartment composition can be the same as described herein for the method of breaking down at least one component of a substrate.

[0067] In various aspects, the catabolic nanocompartment composition can include a liquid coacervate phase including at least one laccase enzyme, and an aqueous phase surrounding the coacervate phase. The composition can degrade lignin to form lignin degradation products when the lignin is contacted with the liquid coacervate phase. In various aspects, when the lignin is contacted with the liquid coacervate phase and the lignin degradation products are formed, repolymerization of the lignin degradation products is decreased and / orprevented compared to exposure of the woody biomass particle to the one or more laccase enzymes outside of the catabolic nanocompartment.Method of making a catabolic nanocompartment composition.

[0068] Various aspects of the present disclosure provide a method of making a catabolic nanocompartment composition. The method can be any suitable method that prepares a composition including the catabolic nanocompartments described herein. The method can include providing an aqueous solution including coacervate-forming components. The method can also include adding at least one catalyst to form a liquid coacervate phase containing the at least one catalyst, to form the catabolic nanocompartment composition. The catabolic nanocompartment composition can degrade at least one component in a substrate to form degradation products of the at least one component when the at least one component is contacted with the liquid coacervate phase. In various aspects, when the at least one component is contacted with the liquid coacervate phase and the degradation products of the at least one component are formed, repolymerization of the degradation products of the at least one component is decreased and / or prevented compared to exposure of the at least one component to the one or more catalysts outside of the catabolic nanocompartment. The catabolic nanocompartment composition can include the same catabolic nanocompartments as described herein for use in the method of breaking down at least one component of a substrate. The components used in the method of making a catabolic nanocompartment composition can be the same as described herein for the method of breaking dow n at least one component of a substrate.

[0069] In various aspects, the method can include providing an aqueous solution including coacervate-forming components. The method can also include adding at least one laccase enzyme to form a liquid coacervate phase containing the laccase enzyme, to form the catabolic nanocompartment composition. The catabolic nanocompartment composition can degrade lignin to form lignin degradation products when the lignin is contacted with the liquid coacervate phase. In various aspects, when the lignin is contacted with the liquid coacervate phase and the lignin degradation products are formed, repolymerization of the lignin degradation products is decreased and / orprevented compared to exposure of the woody biomass particle to the one or more laccase enzymes outside of the catabolic nanocompartment.Examples

[0070] Various embodiments of the present invention can be better understood by reference to the following Examples which are offered by way of illustration. The present invention is not limited to the Examples given herein.Part I. Theoretical basis and proposed methodologies for CatnC-mediated lignin breakdown.

[0071] Decades of effective suppression of naturally occurring fires and increased aridity and weather instability due to extreme weather have resulted in much of the Western U.S. being highly prone to catastrophic wildfire. The primary current tool for wildfire mitigation involves thinning followed by "‘controlled'’ burning of the resulting slash. Unfortunately, such activities have resulted in the ignition of catastrophic mega-wildfires, w ith significant loss of life and property and environmental and economic degradation. There is a significant need for new approaches for disposal of thinned slash, and at the same time, a growing need to replace petroleum-based feedstocks to lessen the greenhouse gases that imperil forest health through extreme weather. Coniferous forests contain significant stores of potential chemical feedstocks that are, in principle, readily converted to valuable products by fermentation and synthetic biological methods. These feedstocks are present in wood in a refractory¬ complex matrix with lignin, which is not readily utilized by existing naturally derived, green industrial processes. While thermochemical methods have been used for lignin degradation to unlock cellulose-based feedstocks from wood, these pose environmental and safety- risks, making them unsustainable. This disclosure describes a groundbreaking new approach toward lignin deconstruction by developing bioinspired, cell-free, catabolic nanocompartments (CatnCs) that are naturally derived and designed to overcome significant mass transfer and product inhibition issues that plague existing biochemical approaches. The development of CatnC technology has required a convergent research approach incorporating several synergistic fundamental advances toprovide a transformative new technology for efficient, economically feasible use of woody biomass.

[0072] Nature has evolved numerous types of liquid polymer-based coacervate materials to control biochemical reactions within and outside cells. For example, recent studies elucidate the way in which such coacervates can maintain functional, extracellular reducing environments within oxidizing milieus while enhancing molecular adhesion and sol ubi 1 ity . The CatnCs described herein can (i) concentrate active oxidases, (ii) solubilize lignin degradation products and redox mediators, and (iii) have interfacial properties necessary to engulf lignin and woody particles effectively.

[0073] Various aspects of the present disclosure will establish a new model for economical forest sustainability and wildfire mitigation by developing highly efficient new pretreatment processes for utilizing woody biomass to enable green, sustainable production of wood-derived chemical products.Various aspects of the present disclosure will help mitigate the enormous ecological, financial and cultural costs of wildfires by eventually providing revenue streams to offset forest health treatment costs. The CatnC platform technology will also enable applications beyond lignin deconstruction, such as decontamination, recycling, and new therapies.

[0074] Goals and Specific Objectives. Thinned slash consists of biomass rich in cellulose and other compounds that are, in principle, valuable precursors for biofuels and commodity / specialty chemicals obtainable through recently developed green biosynthetic methodologies. The primary impediment to (bio)chemical access to these precursors is their encapsulation and close association with lignin, a chemically recalcitrant, heterogeneous macromolecule with little regularity and historically low commercial value. While many methods have been investigated to depolymerize lignin or convert it to value-added products through chemical means (solvents, high temperatures, pressure), these approaches can require (or produce) toxic reagents (or byproducts), obviating the environmental benefits of this renewable bioresource. Various aspects of the present disclosure aim to tackle the “lignin barrier” problem through revolutionary, eco-friendly approaches that will stem from biochemical engineering, synthetic biology, and materials science. Oxidases (specifically laccases) and peroxidases that are known to break down lignin are found innumerous fungi and bacteria, nature’s primary forest product recyclers. A goal of various aspects of the present disclosure is to exploit, engineer, and evolve laccase-based methods to enable efficient, irreversible lignin depolymerization from model coniferous feedstocks that represent the typical makeup of most western thinning waste.

[0075] Objective 1 : Engineering of catabolic nano compartments (CatnCs) for efficient lignin deconstruction. A significant limitation of traditional chemical and enzy matic methods for lignin degradation is the tendency for oxidized lignin subunits to quickly recombine (repolymerize) after they form, hampering breakdown efficiency. This objective has developed means for enveloping wood particles with highly reactive laccase assemblies that will rapidly break down lignin and stabilize oxidative products until they are degraded to solubility'.

[0076] Objective 2: Engineering aqueous media for enhanced lignin degradation. Ionic liquids have recently been shown to enhance the solubility of lignin oxidation products; mixtures of deep eutectic solvents (DES), a class of ionic liquids, and water have been proposed as green alternatives that are both capable of dissolving lignin byproducts and biocompatible. This obj ective explores aqueous DES mixtures compatible with laccase-based CatnCs and capable of efficient solubilization of lignin degradation products.

[0077] Objective 3: Optimization of laccases for efficient lignin oxidation in CatnCs. Once proof of concept demonstration from Objectives 1 and 2 are achieved, components of promising CatnCs can be optimized to increase or maximize chemical reactivity. This can be achieved via combinatorial experimental methodologies using the most efficient natural or engineered laccases in model molecular architectures.

[0078] Lignin in woody biomass has long been considered a nuisance and impediment to utilizing cellulose and hemicellulose in industrial processes, such as pulping and paper making, where it is often simply burned solely for its caloric value. As such, much recent effort has focused on overcoming this “lignin barrier” in the push toward biomass valorization as an alternative to petroleum feedstocks for liquid fuels and commodity and specialty chemicals. Significant progress has been made in developing pretreatment methods for lignin deconstruction (depolymerization) that rely on high temperature, pressure,inorganic catalysis, or bio-incompatible acid or alkaline solvent conditions. While effective, these thermochemical pretreatments can result in toxic waste streams and byproducts that are incompatible with downstream bioprocessing that utilizes biomolecules and cells for the metabolism of cellulosic compounds or biosynthetic conversion of a range of precursors to high-value products. Green routes to lignin breakdown seek to take advantage of molecular mechanisms utilized by organisms that naturally catabolize lignin. Perhaps the most efficient of these are the white-rot fungi (e.g., agaricomycetes, basidiomycetes), which have evolved sophisticated pathways for utilizing wood components, specifically lignin decomposition. While direct utilization of these organisms is not feasible in biomass conversion, substantial effort has been expended in attempts to utilize the evolved lignin-degrading enzymes from these organisms, including laccase and peroxidase enzymes. These catalysts have been used with some success although a significant limitation is the tendency for oxidized lignin to rapidly “repolymerize” after enzymolysis. Various aspects of the present disclosure present a completely new cell-free methodology for the efficient enzymatic catabolism of lignin, while minimizing the repolymerization process.

[0079] Natural metabolic processes allow elegant orchestration of complex reaction networks that have evolved to ensure that forward reactions are favored by precise colocalization of enzymes to rapidly turn reaction products to new7products incapable of reverse reactions. This general strategy has not been employed in designing green, enzyme-based catabolic breakdown of lignin from complex woody feedstocks, such as those derived from small trees and branches from slash piles. Various aspects of the present invention include deployment of cell-free nanoreactors with high oxidase concentrations directed to the lignin to be degraded. These can eliminate both mass transfer limitations to lignin oxidation, as well as opportunities for oxidized lignin to repolymerize. Because optimal molecular design for oxidases within these nanoreactors is not necessarily the same as for those studied thus far in homogeneous aqueous solutions, minimal oxidases have been investigated that have previously been used to form molecular assemblies but do not have as high of a redox potential and substrate turnover rate as the best oxidases available. These minimal oxidases may be better suited to “ganging” in highly concentrated molecularensembles that circumvent mass transfer limitations of enzymes and are optimized for oxygen availability. Simpler, highly tunable, bacterially derived oxidases may be perform better than those evolutionarily optimized to have high redox potential.

[0080] An effective green methodology for surmounting the lignin barrier to unlock the feedstock potential of woody biomass can enable many of the rapidly emerging methodologies for green conversion of cellulosic and other molecular precursors in the biomass to become feasible, generating an economic incentive for forest thinning, enhancing the safety and sustainability of near forest communities. Many of these communities are severely disadvantaged economically, so the benefits can be both environmental and economic.

[0081] Fire is important for maintaining forest health; it remains a natural means to revitalize forest undercarriage to maintain overall forest health. However, numerous, large slash piles resulting from thinning unnaturally overgrown forests represent a significant hazard that, when lit (deliberately or not), can bum for days or weeks while weather conditions are unforeseeable. These slash piles can be targeted as a primary biomass resource (FIGS. 1A-C). Processing them as a feedstock for the creation of valorized products (e.g., fuels, precursors) will offset the costs of thinning as a forest health treatment. This new virtuous cycle toward forest management (along with a premature bum) is shown (FIGS. 1A-C).

[0082] FIG. 2A illustrates a schematic of components of a catabolic nanocompartment (CatnC). The CatnC can further optionally include a redox mediator component (not shown). FIG. 2B illustrates a typical lignin degradation reaction achieved by laccase (note that implementation of redox mediators within the CatnC can enable laccase-based cleavage of phenolic carbon-carbon bonds as well). Phase-separated redox-nanoreactors (CatnCs) can be used for efficient, environmentally friendly deconstruction of lignin in milled particles from woody feedstocks. Design of CatnCs for efficient lignin catabolism can include the simultaneous implementation of several design criteria. 1) To provide effective catalysis, the phase-separated nanoreactors can accommodate a high concentration of oxidase. The nanoreactors can be highly efficient in catabolism because they can enable extremely fast mass transfer of enzymes and redox mediators to the lignin surface. 2) Coacervate-basednanoreactors can have a high concentration of oxygen. 3) The coacervates can effectively solubilize lignin degradation products. Finally, 4) fluid coacervatebased nanoreactors can effectively adhere to and spread over wood and lignin particles to maximize contact of the enzy mes with their lignin substrates.

[0083] An initial model oxidase system used in the development of CatnCs for lignin degradation has been based on the “small laccases." Laccases (EC 1.10.3.2) are a broad class of oxidoreductase enzymes within the multicopper oxidase superfamily that can catalyze the oxidation of a wide range of substituted phenols and many other aromatic compounds, including moieties found in lignin, while reducing molecular oxygen to water. The first laccase was discovered in the Chinese lacquer tree (Toxicodendron vermcifluum) in 1883. and they have since been found in a wide variety of organisms and have been shown to serve a variety of physiological functions. The catalytic site of laccases includes four copper ions, and the electrochemical potential of the enzy me can vary substantially depending on the source, e.g., from 0.4 to 0.8V. The highest values are typically measured for fungal laccases, some of which have evolved to be key players in the degradation of woody lignin. By comparison, plant and bacterial laccases ty pically have lower redox potentials. These redox potentials are sufficient for the oxidation of phenolic residues in lignin; oxidation of other residues can be accomplished through the implementation of low molecular weight, redox mediator compounds such as 1 -hydroxy benzotriazole (HBT), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). One of the most prominent and oxidation-susceptible subunit linkages, in lignin is the / -O-4' linkage. Due to their broad versatility as oxidases and their utilization of molecular oxygen, laccases have been described as the “ultimate green catalysts,” finding utility' in a wide range of industrial and medical applications, such as pulp, paper / textile washing, bleaching / dying, food processing, bioremediation, biosensing, and biofuel cell catalysis. While most laccases are classified as having 3 subunit domains, a 2-domain laccase from Streptomyces coelicolor has been described, which was termed a small laccase (SLAC), and was found to be remarkably active and stable in surfactants, gels, and at high temperatures. The gene for the SLAC was cloned and was successfully expressed heterologously in E. coli, after the activity thereof was documented.

[0084] SLACs were selected for constructing phase-separated CatnCs for several important reasons: 1) high stability; 2) they are readily expressed recombinantly in E. coh and are easy to engineer and test in various molecular contexts; and 3) it is hypothesized that their minimal molecular size compared to other laccase family members and other oxidases will make them ideally suited for inclusion as clients in liquid coacervates and may reduce oxygen mass transfer limitations to the sites of lignin oxidation. The polymer coacervates formed will be liquid aqueous coacervates (vs. crosslinked gels).

[0085] Objective 1 : Engineering of CatnCs for highly efficient lignin deconstruction. This obj ective has developed SLAC-enriched, phase-separated oxidative nanoreactors (CatnCs) that spontaneously coat the surface of woody particles to enable efficient lignin degradation while preventing repolymerization. As there is no precedent to this green approach for enhancing the local concentration of oxidases to enable efficient lignin catabolism, the requisite fundamental components and molecular processes have been developed to achieve each design criterion outlined above. CatnC design criteria can include high concentration of oxidase, high concentration of oxygen, solubilization of lignin products, adhesion to / spreading over wood, and solubilization of mediators / lignin products.

[0086] To form an effective CatnC matrix, a macromolecular coacervate system can be fluid and capable of hosting the requisite molecular clients, including enzymes, reactants, and mediators in their active forms and lignin products in a manner that prevents their repolymerization. Potential simple and complex macromolecular coacervate systems have been examined including intrinsically disordered proteins (IDPs), synthetic polymers, or mixtures of IDPs and synthetic polymers. IDPs have the advantage of being completely compatible (miscible) with similar IDP sequences that can be fused at the genetic level to recombinantly expressed enzymes to introduce “coacervation tags7’ to the SLACs to increase their propensity to partition into the coacervate phase (i.e., become clients or hostages). Several IDPs (natural, recombinant, synthetic) have been investigated in the formation of simple and complex coacervate systems; they are generally more expensive to implement than synthetic polymers so we also investigate synthetic polymers as the likely, ultimate coacervate matrices for the envisioned CatnC technology.

[0087] Several macromolecular systems have been examined that form aqueous coacervate phases, including complementary polyelectrolytes that form complex coacervates and protein and synthetic polymers that form simple coacervate phases (Table 1).

[0088] Table 1. Examples of the aqueous phase-separating solutions examined as matrices for oxidative nanoreactors.

[0089] A first subobj ective is to examine the conditions under which candidate solutions (simple or complex) form liquid coacervates. Each of the solutions listed above can be examined under various conditions of pEI, temperature, and ionic strength. The liquid-like nature of resulting coacervate phases can be examined simply by ejecting them from micropipettes into their supernatant solution and quantifying the time required for them to relax to form spherical droplets either immediately after ejection or upon coalescence. Note that the IDP based simple coacervate systems listed above are not commercially available. These components can be recombinantly expressed in E. coll.

[0090] It is unclear which of the aforementioned coacervate systems are optimal as a basis for oxidative CatnCs; polyelectrolyte-based complex coacervates may be ideal for solubilizing catechol-like lignin products, but they may exhibit lower oxygen solubility than less polar systems. An initial survey can thus be conducted of several aqueous coacervating systems to document the likelihood of serving as reactive compartments that effectively solubilize oxygen, lignin degradation products, and potential redox mediator compounds.

[0091] The solubility of oxygen in each of the aqueous coacervate and supernatant phases (and thus equilibrium partition coefficients) to be examined can be measured using a Clark electrode (oxygen reduction). Depending on the stability of the coacervate phases, measurements can be taken over a temperature range of 15-70°C and a pH range of 4-9. In parallel experiments, equilibrium partition coefficients of model lignin degradation products such as vanillic acid or ferulic acid can also be measured in each candidate coacervate system as afunction of pH and temperature by absorption measurements. For such measurements, serial dilutions of the model lignin degradation compound can be allowed to equilibrate with the phase-separated coacervating system and then decant the supernatant for high throughput measurements in a plate reader. Similarly, the partition coefficients of redox mediators can be documented such as ABTS that have been determined to effectively enable lignin degradation when used in combination with laccase. Once partition measurements for single solutes are achieved, the multi-solute partitioning can be examined in optimal coacervate formulations using the same measurement methodologies. If needed, solubilizers (e.g. cyclodextrins) can be introduced into the coacervation system. Similarly, if poor oxygen solubility is an issue, it may be possible to include materials with high oxygen solubility (e.g., silicone nanoparticles) as oxygen reservoirs into the coacervate systems. Another important characteristic of effective coacervate candidates is the facile diffusion of redox mediators; diffusivities of redox mediators can be measured electrochemically and by fluorescence recovery after photobleaching. As coacervate phases can be modified by adding cosolutes, systematic microscopic examination can also be conducted, including in water-in-oil emulsion droplets that provide a convenient window to visualize coacervate behavior.

[0092] SLACs recombinantly expressed in E. coll can be readily engineered to be fused to a number of ‘'coacervation tags” that exhibit multivalent interactions with macromolecular (protein or polymer) components of complex or simple coacervating systems so that the fusion proteins are incorporated into the coacervate phases as “hostages”. For example, in a polyelectrolyte-based complex coacervate system, a polyanion- (or poly cation-) SLAC fusion protein can be incorporated as part of the overall charge-neutral coacer ate by replacing an equivalent amount of polyanionic (or poly cationic) polymer component in the solution. Similarly, sequences from IDPs that undergo simple coacervation (Table 1) can be expressed as SLAC fusion proteins so that the SLAC is recruited into coacervates formed by the free IDP. Importantly, SLACs fused to IDPs that undergo triggered simple coacervation can be used as a means to rapidly purify the enz mes from cell culture media, which may be a significant benefit to cost-effectively scale up their production. To incorporate SLACs as hostages into coacervate systems formed fromsynthetic polymers (ultimately perhaps the most cost effective alternative), the SLAC can be engineered to incorporate reactive functional groups for sitespecific polymer conjugation, or an IDP based fusion tag can be chosen that is miscible with the synthetic polymer-based coacervate.

[0093] Through these methodologies, SLACs can be incorporated into liquid coacervates, such as those formed from the macromolecular components listed in Table 1. The characteristic absorption spectrum of the cupric metal centers of SLACs can allow quantification of their concentrations in these coacervate phases. Their activity in oxidation of soluble model lignin surrogates (e g., 2,6-dimethoxyphenol, DMP) over a range of conditions (temperature, pH) can be documented by spectroscopic and electrochemical (cyclic voltammetry, rotating disk, impedance spectroscopy) methods. The potential for interaction of various SLAC-containing coacervate phases with suspensions of model woody substrate particles can be examined, and the lignin-degradation process in the presence and absence of redox mediators can be followed via optical microscopy and spectroscopy.

[0094] While liquid coacervates formed from polymeric systems such as those listed in Table 1 have very low interfacial tensions, it is possible that they will not spontaneously stick to and spread over the surface of woody particles. The tendency for liquid coacervate phases to coat a substrate is given by the spreading coefficient (.S’), which is given by the balance of interfacial tensions between the wood particle and the dilute phase or supernatant (ywd), between the wood and the coacervate (ywc), and between coacervate and the dilute phase (yCd).S = d- (ync- Ted)

[0095] Engulfment of the wood particle by the coacervate phase requires that > 0. In the envisioned application, there is no opportunity to modify yw) there is, however, means to reduce the other two interfacial tensions (y«.. and ycd) if necessary to enhance the tendency for coacervates to stick to wood particles in suspension and to spread over, and engulf them. As mentioned above, ycd is generally low for liquid coacervates such as those formed from the macromolecular systems (Table 1). It can be further reduced by spiking the macromolecules in solution with block copolymers of the coacervating polymer and a surface active (e.g., highly water soluble) block. Further, it ishypothesized that ywccan be decreased by introducing a molecular recognition element that binds specifically to a component of the woody particle.Specifically, conjugation of known lignin binding domains and cellulose binding domains into coacervate-forming proteins and polymers can enhance coacervate adhesion and spreading onto woody particles.

[0096] Microscopic examinations can be conducted of the extent of the spreading of simple and complex coacervates over woody particles as a function of solution conditions (temperature, pH). Fluorescently labeled coacervate components can be employed to enhance imaging contrast. Diffusivities of various coacervate components (polymers, clients, and hostages) can also be characterized by fluorescence recovery after photobleaching (FRAP) measurements. Transport properties (coacervate viscosity and diffusivities) may be influenced by temperature, due to the potential sensitive dependence of equilibrium polymer volume fraction in the coacervate on temperature. As indicated above, water-in-oil emulsion droplets produced by microfluidics can be used as windows to allow facile visualization of coacervate properties.

[0097] After formation of laccase-containing CatnCs that effectively oxidize soluble lignin analogues (e.g., 2,6 DMP, guaiacylglycerol-P-guaiacyl ether, GGE. a dimeric lignin analogue), their suitability can be tested for degradation of model technical lignins and model coniferous wood particles. Optimization of conditions for lignin depolymerization using coacervate-based CatnCs can be conducted in highly parallel experimentation utilizing microtiter plates, a plate reader, and droplet arrays. For example, suspensions of lignin or wood particles and the model oxidative coacervates can be incubated in a filter microplate for a given amount of time, after which the supernatant can be transferred to a reading plate for analysis. Initial experiments can be conducted with minimal coacervate formulations (polymers, conjugated SNACs), and as necessary, more complexity can be incorporated to the formulations using the various components described above (redox mediators, oxygen reservoirs, spreading promotors). In parallel to these suspension-based ensemble measurements, detailed microscopic characterization can be conducted of the structure and reactivity of the CatnCs, using optical microscopy and droplet arrays, along with more advanced methodologies.

[0098] Objective 2: Engineering aqueous media for enhanced lignin degradation. Ionic liquids (ILs) have been extensively studied in a wide range of industrial processes, including biorefining, because of their excellent solvating capabilities, low vapor pressures, and high stability. A number of IL systems have been used to dissolve biomass and lignin degradation products in biofuel production. Biomass-derived ILs have also been demonstrated for biorefinery applications, pointing to an important means for self-sustenance of biomass utilization processes. In green, biological approaches to lignin deconstruction, many ILs have destabilizing effects on oxidative enzymes. There has been significant effort to identify / select ILs and enzy me variants with enhanced stability and reactivity. In principle, solvent composition can have favorable effects on reaction equilibrium and minimize the production of unwanted byproducts.

[0099] Deep eutectic solvents (DESs) represent a variation on ILs that have been investigated as specifically well-suited for enzymatic degradation of lignin because they exhibit biodegradability, biocompatibility, and low cost compared to other ILs. DESs consist of a hydrogen bond donor (e.g., ethylene glycol, glycerol, ery thritol, xylitol, or propionic acid) and a hydrogen bond acceptor (e.g., choline chloride, choline dihydrogen citrate, or betaine) in integral ratios. They can show high solubility for lignin and compatibility with laccase and other lignin-degrading enzymes.

[0100] It is hypothesized that optimization of DES / aqueous solvent systems in which to deploy the CatnCs facilitates enzymatic access and activity to lignin in woody biomass while at the same time better accommodating lignin degradation products to minimize repolymerization. The activities of SLACs and SLAC fusion proteins in various DES / aqueous mixtures can be tested.Systematically examination of IDP and synthetic polymer coacervation (simple and complex) in DES / aqueous mixtures can be performed. Given the enhanced solvent properties of these mixtures, modification of the sequence of coacervating polymers and proteins (e.g., to make them less water soluble) may7be needed to obtain coacervates under conditions commensurate with high laccase activity. Once laccase activity and coacervate studies point to suitable conditions for the formation of CatnCs, laccase fusion proteins can be combined with coacervate components and the performance of CatnCs using progressivelymore complex substrates (e.g., lignin surrogates, technical lignin, woody biomass) can be tested as described in Objective 1.

[0101] Objective 3: Optimization of laccases for efficient lignin oxidation in CatnCs. Because of their tremendous industrial importance as green catalysts, many laccase t pes from fungi, plants and bacteria, have been cloned, heterologously expressed, and characterized. Laccases have also been the subject of numerous protein engineering studies in which various methodologies for rational / semi-rational design and directed evolution have been implemented. Several methods for achieving secretion and surface display of recombinant laccases have been developed, thus enabling direct lignin degradation without protein purification, and importantly, along with efficient high throughput screening of their activities and genetic encoding for selection of mutants with desired properties. As a result, laccases with a wide range of properties have been generated, including variants that are stable and active at high temperatures and in unnatural media (e.g., acid / alkaline conditions, ionic liquids, physiological fluids). The engineering and directed evolution of SLACs has been limited, probably because of their relatively low redox potentials.Likewise, laccase engineering or directed evolution has not yet yielded variants that are selected for enhanced stability and high activity in DESs, though this will probably be achieved in the near future.

[0102] SLACs are initially chosen as a model system for development of CatnCs because they are small relative to 3-domain laccases, highly stable, have been recombinantly expressed as fusion proteins that allow them to assemble into crosslinked gel architectures, and because in these architectures, they have been shown to outperform non-crosslinked enzymes that are more complex and have higher redox potentials. However, there is no reason to assume that SLACs are the optimal enzy mes for implementing CatnCs for the degradation of lignin from woody biomass so we can examine the performance of a range of bacterial and fungal enzymes as active hostages in CatnCs. Incorporation of most fungal enzymes, many of which incorporate glycosylation, can require recombinant expression in fungal cell lines (e.g., in yeast, e.g., Pichia pastoris, Saccharomyces cerevisiae).

[0103] As a first step to examine the possible incorporation of 3-domain laccases (e.g., non-SLACs) into CatnCs, a limited set of well-characterized,heterologously expressed bacterial and fungal laccases can be chosen based on several factors, including their documented expression yield, redox potential, thermal and aqueous stabilities, and previous investigation in lignin depolymerization. Plausible candidate laccases include commercially available laccase from Trametes versicolor, CotA from Bacillus subtilis (expressed in E. coll), laccase from Myceliophthora thermophilia (expressed in S. cerevisiae), laccase 420 from Trametes sp. (expressed in P. pastoris), laccases engineered by site-directed mutagenesis (e.g., CueO from E. coll and CotA) or saturation mutagenesis (e.g., from M. thermophilia), and laccases selected for tolerance to ionic liquids by directed evolution (e g., Lcc2 from T. versicolor . Of particular interest are a highly thermostable, high redox potential laccases (HRPLs).Laccases from basidiomycetes fungi can exhibit relatively high redox potentials (~0.8 V), allowing them to degrade a broad range of compounds that other laccases cannot oxidize, yet compared to bacterial laccases, they can have limited thermal stability . The HRPL expressed in, and secreted from, S. cerevisiae, was characterized as OB-1. The thermal stability of OB-1 has been enhanced to evolve an HRPL mutant, termed Lac-Vader, that is readily produced in S. cerevisiae, and that shows excellent stability at high temperatures (e.g., to 75 °C) and at high and low pH.

[0104] Selected 3-domain laccases and their fusions with coacervate tags can be expressed and their activity can be characterized in solution, engineered coacervates, and aqueous solvents as described in Objectives 1 and 2. It is hypothesized that laccases with high redox potentials and high thermal stability can allow enhanced degradation of lignin in model systems and woody biomass.

[0105] The chemical recalcitrance of lignin has long been a problem in pulp and paper manufacture, biorefining, and recycling. Green routes toward lignin deconstruction that utilize microbial or enzymatic digestion have made significant progress but are not yet economically competitive with less sustainable chemical means. The primary outcome and impact of the various aspects of the present disclosure is a new- green platform technology for lignin deconstruction that addresses fundamental limitations associated with mass transport, cataly st activity and product solubility. While we are focused on utilization of waste generated in the maintenance of forest health, the platform technology disclosed herein can have numerous applications across biomassutilization (e.g., for processing switchgrass, com stover and cobs, cardboard, paper, and the like) for the production of a wide variety of value-added commodity and specialty chemicals. Processing thinned slash can have a significant economic offset in the economic feasibility associated with the cost of preventative thinning and prescribed burning (currently ~$2-4K / acre). meaning any profit made by utilizing thinned biomass can offset the costs necessary to prevent catastrophic wildfires. Aside from the primary benefits of various aspects of the present disclosure, other positive outcomes can be associated w ith the secondary' concepts to be developed, including (i) highly reactive laccase-based coacervates, (ii) engineered coacervate-based redox nanoreactors, (iii) aqueous DES-based reactive coacervates, and (iv) modified laccases for function in CatnCs and DES / aqueous mixtures, which can address issues of stainability, extreme w eather, and health.

[0106] Various aspects of the present disclosure have several positive societal implications. Development of competitive, green enzymatic technologies for lignin deconstruction can circumvent the use of toxic and dangerous (high temperature, high pressure) conditions associated with chemical methods. Various aspects of the present invention avoid the implementation of genetically modified organisms (GMOs) in biorefmery settings, and the GMOs used to generate the recombinant enzymes and polymers to be used can be contained in centralized facilities under well-established, strict biosafety¬ protocols. Various aspects of the present disclosure allow profitable utilization of thinned biomass (slash) that would otherwise simply be burned in the field, creating a significant risk for catastrophic wildfires that have resulted in tremendous loss of property', livelihoods, watershed health, human health, agricultural productivity, and community' and cultural cohesion.

[0107] Various aspects of the present disclosure represent a significant shift in fundamental engineering approaches for biomass utilization and biorefining. Various aspects of the present disclosure can provide a new paradigm for reducing mass transfer limitations by increasing enzymatic access to substrates, allowing for efficient removal of reaction products. The development of CatnCs for the challenge of lignin deconstruction can provide a platform technology for the pretreatment of hgnin-containing biomass well beyond coniferous woody waste to several other biomass feedstocks. The keystep of lignin deconstruction can enable the success of many promising yet still challenging processes for biomass utilization, including the production of engineered lumber, biofuels, bioplastics and textiles, pharmaceuticals, commodity chemicals, and other valuable natural products. While the focus here is on lignin deconstruction, a key limiting process for utilization of woody biomass, this general technology of coacervate-based enzymatic nanoreactors can find utility in a number of other biochemical conversions, including biofuel cell catalysis, detoxification, bioremediation, disinfection, and enzymatic therapies. Various aspects of the present disclosure can lead to a range of powerful technologies with substantial long-term impacts beyond biomass utilization and will be driven by the urgent need to address the grand challenges of sustaining natural and agricultural environments and developing alternatives to fossil fuels and feedstocks.Part II. Experimental demonstration of CatnC formation and function.Example II-A, LB-ELP coacervates coat lignin particles.

[0108] This Example tests the hypothesis that LB2-80 (lignin-binding elastin-like polypeptide (ELP)) can bind to and coat lignin particles upon LLPS and coacervation.

[0109] Materials and Reagents. Kraft lignin (Sigma-Aldrich): resuspended in IX phosphate-buffered saline (PBS). AF488-LB2-80: Alexa Fluor 488 fluorescently labeled LB2-80.

[0110] Microscopy of Microdroplets. Suspensions of kraft lignin were created by dispersing it in IX PBS and subjecting the suspension to multiple filtering steps to obtain suspensions with particles with sizes below 10 microns. The lignin suspension was mixed with an equal volume of LB2-80, resulting in a final LB2-80 concentration of 0.5 mM. LB2-80 was mixed in a ratio of 1:5 AF-488-labeled LB2-80 to unlabeled LB2-80. The sample was emulsified in mineral oil containing 3% (v / v) Abil EM 90 surfactant to produce stable aqueous microdroplets suitable for microscopy. Images were taken as the temperature was increased from 10 °C (below the phase transition temperature) to 45 °C, which is above the phase transition temperature. Additional images were taken periodically as LLPS occurred, monitoring the progression of coacervation.Samples were imaged on an Olympus 1X83 epifluorescence microscope using the corresponding filter sets for AF488 and Cy3.Discussion.

[0111] FIG. 3 illustrates fluorescence microscopy images of aqueous microdroplets containing lignin particles and LB2-80 and the temperaturedependent LLPS of AF488-labeled LB2-80 and its spatial association with kraft lignin particles, which autofluoresce in the microscope’s Cy3 channel. Upon heating to 45 °C, LB2-80 exhibits coalescence and increased localization of lignin particles.

[0112] At 10 °C, well below the phase transition temperature of LB2-80, the emulsified lignin-protein mixture appeared uniform, with no visible separation or aggregation. Within the aqueous microdroplets, lignin particles were evenly dispersed, and fluorescence imaging showed that AF488-labeled LB2-80 was homogeneously distributed throughout the droplet volume. The dispersed fluorescence pattern indicated that the protein was fully solvated and did not associate with lignin particles, which displayed autofluorescence when imaged through Cy3 filters.

[0113] As the temperature was increased to 45 °C, exceeding the transition temperature of LB2-80, the mixture underwent LLPS. Within minutes, distinct condensed domains became visible, indicating formation of a dense, protein-rich coacervate phase within the aqueous microdroplets. Over approximately 40 minutes at elevated temperature, these domains coalesced into larger, more stable droplets that preferentially accumulated lignin particles. The fluorescence intensify corresponding to LB2-80 increased markedly at the lignin interfaces, producing a sharply defined boundary pattern consistent with physical association of the coacervate phase with lignin particles.

[0114] This temperature-induced reorganization demonstrates that LB2-80 transitions from a uniformly single phase to a condensed phase that selectively partitions to lignin interfaces and envelops lignin particles. The observed accumulation of lignin particles in the LB2-80 coacervate supports the hypothesis that its lignin-binding domain mediates interfacial association upon coacervation. These results collectively indicate that phase separation enhances the spatial proximity between LB2-80 and lignin, promoting the formation of aprotein-enriched coacervate layer at the solid-liquid boundary under conditions supporting phase separation.Example II-B, LB-ELP coacervates coat wood particles.

[0115] This Example tests the hy pothesis that LB2-80 binds to and coats wood particles upon LLPS and coacervation.

[0116] Materials and reagents. Wood particles (ponderosa pine): resuspended in IX PBS. AF488-LB2-80: Alexa Fluor 488 fluorescently labeled LB2-80.

[0117] Microscopy of microdroplets. The suspension of wood particles was created by grinding ponderosa pine, suspending the particles in IX PBS and filtering down to sizes below 10 microns. LB2-80 was mixed in a ratio of 1 :5 AF-488 labeled LB2-80 to unlabeled LB2-80. LB2-80 was added to a final concentration of 0.5 mM. The sample was emulsified in mineral oil containing 3 % (v / v) Abil EM 90 surfactant to produce stable aqueous microdroplets suitable for microscopy. Preliminary images were taken before the temperature was increased to 60 °C, which is above the transition temperature. Additional images w ere taken periodically as LLPS occurred, monitoring the progression of coacervation. Samples were imaged on an Olympus 1X83 epifluorescence microscope using the corresponding filter sets for AF488 and Cy3.Discussion.

[0118] FIG. 4 illustrates fluorescence microscopy images showing temperature-dependent LLPS of AF488-labeled LB2-80 and its spatial association with wood particles, which autofluoresce in the microscope’s Cy3 channel. Upon heating to 60 °C, LB2-80 exhibits coalescence and increased localization of wood particles.

[0119] At temperatures below the transition temperature (e.g., 10 °C), the protein suspension appeared homogeneous, with dispersed fine wood particles and no evidence of phase separation. Fluorescence imaging confirmed that AF488-labeled LB2-80 was uniformly distributed within the aqueous droplets, exhibiting a diffuse signal characteristic of a fully solvated state. The wood particles, taken from ponderosa pine and reduced to sub-10 pm fragments, displayed autofluorescence under Cy3 filters, allowing clear differentiation fromthe protein signal. At this stage, no preferential accumulation of LB2-80 near wood particles was evident.

[0120] Upon heating to 60 °C, the sample underwent LLPS, producing distinct LB2-80-rich coacervate droplets within each aqueous microdroplet. These condensed domains increased in size and brightness over time, consistent with droplet coalescence and maturation of the coacervate phase. After approximately 25 minutes at elevated temperature, the protein-rich droplets were observed to localize preferentially at the wood particles. The fluorescence signal intensified with time, producing visible ring-like patterns around the solid wood fragments, indicative of adsorption or coating of the wood surfaces by the condensed LB2-80 phase.

[0121] This temperature-driven localization behavior reveals that LB2-80 retains strong affinity for lignocellulosic substrates even within complex composite materials such as wood. Unlike the kraft lignin system, the w ood particles present chemically and structurally heterogeneous surfaces that include lignin, cellulose, and hemicellulose domains. The selective enrichment of LB2-80 at these interfaces suggests that its lignin-binding domain drives adsorption to lignin-rich regions, while the coacervate state enhances interfacial residence by concentrating the protein at the solid-liquid boundary. Together, these observations demonstrate that LLPS facilitates spatial targeting of LB2-80 to natural lignocellulosic interfaces, enabling the formation of a condensed, polymer-rich layer surrounding wood particles under coacervating conditions.Example III. Binding of LB2-80 to lignin.Example III- A, Lignin-binding assay: electrophoretic retardation.

[0122] It is possible to solubilize kraft lignin by suspending it in 0.1 M NaOH. The resulting solution can be added to buffers compatible with laccase activity. Our hypothesis is that gel electrophoresis can be used to verily that such solubilized lignin is bound by LB2-80 in its soluble form.

[0123] A lignin retardation assay was developed to determine the interaction betw een lignin and a lignin-binding protein (LB2-80) by monitoring the migration of lignin during agarose gel electrophoresis. The method is conceptually analogous to DNA-protein retardation assays, in which formationof macromolecular complexes reduces the electrophoretic mobility of the charged substrate. Under alkaline conditions, lignin behaves as a poly anion due to deprotonation of phenolic groups and migrates toward the anode. When LB2-80 binds to lignin, leading to measurable retardation in migration through the agarose matrix. The degree of retardation is proportional to the concentration of the protein, thereby serving as a functional measure of lignin-protein interaction.

[0124] Agarose gels (1% w / v) were prepared by dissolving molecular-biology-grade agarose in 36 mM sodium borate buffer (SB, pH 8.8). The molten gel was cooled to 60 °C and cast in a gel tray with 8 x 2 mm wells. After solidification, the gel was submerged in the same buffer.

[0125] Each loading mixture contained 10 pL of sample and 4 pL of 50% glycerol. Ten microliters of each mixture were loaded per lane. The gel was run at 50 V for 90 min using a Bio-Rad PowerPac Basic power supply.

[0126] Gels were visualized using a ChemiDoc (Bio-Rad) imaging system equipped with a dual-color tray and SYBR Safe filter set. The standard 590 / 110 nm filter (broad orange emission) was employed under visible illumination mode used for protein and DNA gels and blots.

[0127] Lignin exhibits intrinsic fluorescence in the visible range, and its emission intensity’ is directly correlated with lignin concentration within the gel.

[0128] Kraft lignin (Sigma- Aldrich) was dissolved in 0.1 M sodium hydroxide to a concentration of 10 mg ml and then diluted to 5 mg mL1in eight individual samples. Seven of these samples additionally contained the fusion protein LB2-80, serially diluted to final concentrations of 500, 100, 50.10, 1, 0.1, and 0.01 pM. One sample containing lignin alone served as a negative control.

[0129] All mixtures were gently vortexed, incubated for 1 hour at room temperature, and then loaded directly into the gels. Gel composition, running conditions and gel visualization were previously described.Discussion.

[0130] FIG. 5 illustrates agarose gel electrophoresis showing concentration-dependent retardation of lignin migration by the LB2-80 protein. Decreasing LB2-80 concentrations (lanes 2-8) progressively increase lignin mobility' compared to the lignin-only^ control (lane 1)

[0131] Observed migration patern (FIG. 5):

[0132] Lane 1 (lignin only, 0 pM LB2-80): A single fluorescent band migrates away from the well (baseline mobility).

[0133] Lanes 2-3 (500, 100 pM LB2-80): Fluorescence is concentrated in the well; litle to no migrating band is visible. This indicated that the majority7of lignin was incorporated into large protein-lignin complexes that were unable to penetrate the gel matrix.

[0134] Lanes 4-5 (50, 10 pM LB2-80): A migrating band is present but travels less than the control in Lane 1 (partial retardation). This demonstrates that the complete retardation of lignin mobility by LB2-80 was lost at these low LB2-80 concentrations.

[0135] Lanes 6-8 (1, 0.1, 0.01 pM LB2-80): The lignin band migrates to approximately the same position as Lane 1 (no evident retardation).

[0136] These results establish that lignin migration is inversely proportional to LB2-80 concentration up to approximately 10 pM, consistent with the formation of lignin-protein complexes that alter both the hydrodynamic size and net charge of lignin. The strong retention observed at high protein concentrations reflects the formation of multimeric or cross-linked aggregates that remain trapped in the well. As the concentration of LB2-80 decreases, the complexes become smaller and less stable, permiting partial entry7into the gel and slower migration. Below7approximately 1 pM LB2-80, the equilibrium favors dissociation, and the complexes revert to free lignin species that migrate identically to the control. Dissociation is likely to occur during sample equilibration in the well or early in migration through the gel, when dilution in running buffer and electrophoretic strain reduce the effective affinity of the interaction. The observed concentration-dependent retardation therefore provides direct evidence of binding between LB2-80 and lignin under the assay conditions.Example III-B. Spectroscopic assay of partitioning of solubilized lignin.

[0137] This Example tests the hypothesis that solubilized kraft lignin partitions into LB2-80 coacervates upon LLPS.

[0138] Materials and reagents. Kraft lignin (Sigma-Aldrich): dissolved in 0.1 M NaOH to 1 mg mL LB2-80 (lignin-binding ELP): dissolved in IXPBS to 2 mM. El-40 ([VPGVG]40; non-binding control ELP): dissolved in IX PBS to 2 mM.

[0139] Partitioning assay. For LB2-80 and El -40, separate tubes were prepared with solubilized lignin in NaOH, diluted in IX PBS and 50 mM sodium acetate buffer to a final concentration of 0.05 mg mLBoth ELPs were at a final concentration of 0.5 mM. Initial bulk extinction (which reflects both absorbance and light scattering) was measured at 330 nm. LLPS was induced by allowing the samples to incubate at 40 °C for 1 hour before separating the coacervate and supernatant fractions. The supernatant was withdrawn and read. The coacervate was resuspended to the original volume in the same buffer and read.Discussion.

[0140] FIG. 6 illustrates extinction measurements at 330 nm showing solubilized lignin before phase separation (control) and after partitioning between the coacervate (pellet) and supernatant fractions for El -40 and LB2-80 in sodium acetate and PBS buffers. LB2-80 retained more lignin within the coacervate phase than El-40, especially in PBS.

[0141] Extinction measurements at 330 nm revealed clear differences in lignin partitioning between the lignin-binding ELP (LB2-80) and the nonbinding control (El -40). Prior to phase separation, extinction values were high for all samples, indicating uniform dispersion of lignin in solution. Following LLPS at 40 °C, samples containing LB2-80 displayed a pronounced redistribution of lignin between the coacervate and supernatant fractions.Extinction increased in the coacervate fraction while decreasing in the supernatant, demonstrating preferential sequestration of lignin into the condensed protein-rich phase. This effect was more pronounced in sodium acetate than in PBS, suggesting that ionic environment and pH modulate the extent of lignin-protein association.

[0142] In control samples containing El -40, there was no evidence of consistent selective lignin recruitment: the PBS supernatant remained higher than the pellet, while the opposite was observed for sodium acetate buffer. These results suggest that molecular recognition — conferred by the ligninbinding domain — drives lignin enrichment within LB2-80 coacervates.Incorporation of the lignin-binding domain enables LB2-80 to selectively concentrate solubilized lignin within the condensed phase, distinguishing its behavior from non-binding ELPs and supporting its role as a targeted sequestration scaffold under coacervating conditions.Example IV. LB-ELP coacervates take up laccase clients.

[0143] This Example tests the hypothesis that T. versicolor laccase and SLAC1 partition into LB2-80 coacervates upon LLPS. We observe partitioning in droplets and corroborate enzyme partitioning in bulk fluorescence measurements. El-40 serves as a control. The sequence of SLAC1 is MDRRGFNRRVLLGGAAAATSLSIAPEVAGAAPAAKGITARTAPAGGEV RHLKMYAEKLADGQMGYGFEKGKASVPGPLIEVNEGDTLHIEFTNTMD VRASLHVHGLDYEISSDGTAMNKSDVEPGGTRTYTWRTHKPGRRDDGT WRPGSAGYWHYHDHVVGTEHGTGGIRNGLYGPVIVRRKGDVLPDATH TIVFNDMTINNRKPHTGPDFEATVGDRVEIVMITHGEYYHTFHMHGHR WADNRTGILTGPDDPSRVIDNKITGPADSFGFQIIAGEGVGAGAWMYHC HVQSHSDMGMVGLFLVKKPDGTIPGYEPHEHGGATAKSGESGEPTGGA AAHEHEHGY.

[0144] Materials and reagents. LB2-80: dissolved in IX PBS. El-40: dissolved in IX PBS. Laccase from Trametes versicolor (>10 U mg '): prepared as stock solutions in IX PBS and 50 mM sodium acetate buffer (pH 5.0).SLAC1: a small laccase, prepared as stock solutions in IX PBS and 50 mM sodium acetate buffer (pH 5.0). Kraft lignin (Sigma- Aldrich): suspended in 50 mM sodium acetate (pH 5.0).

[0145] Microscopy of microdroplets. Two types of samples were generated: (1) 0.5 mM ELP with Alexa Fluor 488-labeled SLAC1 (AF488-SLAC1) (5.5 pM) and (2) 0.5 mM ELP with Cy3-labeled T. versicolor laccase (Cy3-laccase) (5 pM). Each mixture was emulsified in mineral oil containing 3 % (v / v) Abil EM 90 surfactant to produce stable aqueous microdroplets suitable for microscopy. LLPS was induced by incubating the emulsions at 40 °C for 30 min to allow formation of coacervate droplets within the aqueous phase.Samples were imaged on an Olympus 1X83 epifluorescence microscope using the corresponding filter sets for AF488 and Cy3.

[0146] Fluorimetry partitioning assay. For LB2-80 and El -40, separate tubes were prepared with Cy3-Laccase in IX PBS and 50 mM sodium acetate buffer at a final concentration of 1 pM. Both ELPs were at a final concentration of 0.5 mM. Initial bulk fluorescence was measured at 530 / 570 nm. LLPS was induced by allowing the samples to incubate at 40 °C for 1 hour before separating the coacervate and supernatant fractions. The supernatant was withdrawn and read. The coacervate was resuspended to the original volume in the same buffer and read.Discussion.

[0147] FIG. 7 illustrates fluorescence microscopy images showing colocalization of LB2-80 coacervates with T. versicolor laccase (labeled with Cy3) and SLAC1 (labeled with AF488). Both enzymes exhibit strong fluorescence within the coacervate phase, indicating preferential partitioning into LB2-80 coacervates.

[0148] Fluorescence microscopy revealed that, following phase separation of LB2-80 above its transition temperature, both T. versicolor laccase and SLAC 1 localized strongly within the condensed coacervate phase. Bright fluorescence from labeled enzymes was observed within the LB2-80 droplets, while the surrounding dilute phase exhibited diminished signal. This colocalization demonstrates that the enzymes partition preferentially into the protein-rich domains formed during LLPS. The strong confinement of enzy me fluorescence within the condensed phase indicates that LB2-80 can act as a reactive compartment, concentrating oxidative enzymes within the coacervate environment. These qualitative observations establish that both laccases are compatible with the LB2-80 coacervate environment and suggest potential for enzy me encapsulation through specific or nonspecific interactions during phase separation.

[0149] FIG. 8 illustrates fluorescence measurements at 530 / 570 nm showing Cy-3 labeled T. versicolor laccase before (control) and after partitioning between the coacervate (pellet) and supernatant fractions for El -40 and LB2-80 in sodium acetate and PBS buffers. LB2-80 retained more laccase within the coacervate phase than El-40, especially in PBS.

[0150] Bulk fluorescence measurements corroborated the microscopy findings of enzyme partitioning. Across both buffer systems, LB2-80 retained more T. versicolor laccase fluorescence in the coacervate fraction than the nonbinding control El-40. In PBS, the coacervate phase of LB2-80 accounted for most of the total fluorescence signal, indicating strong recruitment of enzyme into the condensed phase. In sodium acetate buffer, enzyme enrichment within the LB2-80 coacervate was also evident but to a lesser degree, consistent with the influence of buffer composition and ionic strength on coacervate-protein interactions.

[0151] For El-40, the coacervate fraction contained comparatively little fluorescence, with most enzyme signal remaining in the supernatant. This pattern shows that phase separation alone does not promote enzyme sequestration and that inclusion of the lignin-binding domain in LB2-80 enhances enzy me uptake. Together, these results demonstrate that LB2-80 effectively concentrates laccase clients within its condensed phase, supporting its potential role as a catalytic host material for oxidative reactions involving lignocellulosic substrates.Example V. ABTS partitions into LB-ELP coacervates.

[0152] Hypothesis: the redox mediator ABTS partitions into LB2-80 coacervates upon phase separation. Partitioning was evaluated by measuring the distribution of ABTS between the coacervate and supernatant phases prior to enzy matic oxidation.

[0153] Materials and reagents. Laccase from Trametes versicolor (>10 prepared as a 20 pM stock solution in 50 mM Sodium acetate buffer (pH 5.0). ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): prepared as a 10 mM stock solution in 50 mM sodium acetate buffer (pH 5.0). LB2-80: an elastin-like polypeptide containing a lignin-binding domain, dissolved in IX PBS and added to final concentrations of 0.5 mM.

[0154] Partitioning assay. LB2-80 was prepared at a concentration of 1 mM in IX PBS and added to a final concentration of 0.5 mM. ABTS was prepared at a concentration of 2 mM in 50 mM sodium acetate buffer (pH 5.0) and added to a final concentration of 0.5 mM. LLPS was induced by incubating the samples at 40 °C for 30 min, a temperature above the transition temperature(Tt). LLPS was induced by allowing the samples to incubate at 40 °C for 1 hour before separating the coacervate and supernatant fractions. The coacervate was resuspended to the original volume in the same buffer. ABTS partitioning was characterized by UV-vis spectroscopy prior to oxidation, measuring the characteristic extinction of the reduced ABTS form at 350 nm using an Agilent Synergy Hl microplate reader. The extinction of each fraction (supernatant and resuspended coacervate) was recorded. The total extinction of both fractions was compared to the pre-phase-separation sample to confirm quantitative recovery'.

[0155] Colorimetric assay. Reactions were performed in 1.5 mL microcentrifuge tubes with a total volume of 200 pL. The following compositions were prepared in triplicate for each condition: ABTS (0.5 mM)+ laccase (2 pM), and ABTS + laccase + LB2-80 (0.5 mM). All reactions were incubated at room temperature, above the transition temperature (Tt) of LB2-80, to induce coacervation, for 10 hours. Initial measurements (time Oh) were collected immediately after mixing and analyzed alongside the 10-hour timepoints. Enzymatic oxidation of ABTS was monitored by UV-Vis spectroscopy using an Agilent Synergy' Hl microplate reader. For laccase + ABTS reactions, extinction was recorded at 420 nm, corresponding to the formation of the ABTS+» radical cation. The increase in extinction at this wavelength directly reflects laccase turnover. Degradation was expressed as a relative change in extinction between the 0-hour and 10-hour samples. At the end of the 10-hour incubation, photographs w ere taken to visually assess color changes in the reaction mixtures. Images were captured to compare the color intensity of the solutions containing different combinations of laccase, ABTS, and LB2-80, thereby reflecting the extent of ABTS oxidation.Discussion.

[0156] FIG. 9 illustrates extinction measurements at 340 nm showing before (control) and after ABTS partitioning between the coacervate (pellet) and supernatant fractions for LB2-80 in sodium acetate. Strong partitioning is observed in the LB2-80 coacervate.

[0157] Spectroscopic analysis showed that the redox mediator ABTS strongly partitioned into LB2-80 coacervates following phase separation.Extinction at 340 nm, characteristic of the reduced form of ABTS, was measured in the initial homogeneous mixture, the resuspended coacervate fraction, and the supernatant obtained after LLPS and gentle centrifugation.

[0158] After phase separation, the coacervate fraction retained nearly the full extinction observed in the pre-LLPS control, while the supernatant displayed only a small residual signal. This distribution demonstrates that ABTS was almost completely sequestered within the condensed LB2-80 phase, with minimal mediator remaining in the surrounding aqueous phase. Such preferential partitioning suggests that the microenvironment of the coacervate provides favorable interactions for small aromatic molecules like ABTS.

[0159] Confinement of redox mediators within the same dense phase as enzymes is expected to enhance catalytic turnover by increasing local concentrations and facilitating efficient electron transfer. These findings therefore indicate that LB2-80 coacervates can act not only as enzy me-binding compartments but also as selective reservoirs for small-molecule cofactors, positioning them as promising microenvironments for enzymatic reactions.

[0160] FIG. 10A illustrates quantitative analysis of ABTS oxidation with laccase in the presence of LB2-80 as measured by extinction at 420 nm. FIG.10B illustrates photographs showing macroscopic appearance of laccase-ABTS reactions with LB2-80 after 10 h incubation above Tt in microcentrifuge tubes. FIG. IOC illustrates photographs showing macroscopic appearance of laccase-ABTS reactions with LB2-80 after 10 h incubation above Tt in microtiter wells. Although not visible in FIGS. 10B-C, in the original color photographs, FIGS.10B-C for -LB2-80 and +0.0 ImM LB2-80 (T<Tt) (the first and last photographs) were blue and FIGS. 10B-C for +0.5 mM LB2-80 (T>Tt) and +0.1 mM LB2-80 (T>Tt) (the second and third photographs) were purple.

[0161] Reactions containing laccase and ABTS displayed the characteristic extinction increase at 420 nm, corresponding to formation of the oxidized ABTS,+radical cation. Inclusion of LB2-80 amplified this signal in a concentration-dependent manner, reaching its highest value at 0.5 mM LB2-80. The elevated extinction at 420 nm observed at 0 h for the 0.5 mM LB2-80 sample results from light scattering caused by LB2-80 undergoing LLPS rather than from true color development (FIG. 10A). Therefore, the enhanced enzymatic activity associated with LB2-80 was inferred based on the visualmacroscopic appearance of the samples (FIGS. 10B-C) and the increased 420 nm extinction values measured after 10 h of incubation (FIG. 10A). This trend indicates that LB2-80 enhances laccase catalytic efficiency, likely by increasing local concentrations of both enz me and mediator. The coacervate phase may also promote more effective electron transfer by creating favorable local dielectric properties. The spectroscopic and visual evidence demonstrate that the presence of LB2-80 above its transition temperature markedly influences the rate of ABTS oxidation. By concentrating reactive components within the coacervate phase, LB2-80 enables higher enzymatic turnover, producing a visibly clearer and chemically more oxidized product mixture.Example VI. Formation of CatnCs.Example VI-A, CatnCs: Polymer coacervates that coat lignin and incorporate enzvme clients.

[0162] This Example tests the hypothesis that LB2-80 coacervates will coat lignin particles and recruit laccase, concentrating enzymes in the vicinity of the lignin interface.

[0163] Materials and reagents. AF488-LB2-80: Alexa Fluor 488 fluorescently labeled LB2-80. El-40: dissolved in IX PBS. Laccase from Trametes versicolor (>10 U mg '): prepared as stock solutions in IX PBS and 50 mM sodium acetate buffer (pH 5.0). Kraft lignin (Sigma- Aldrich): suspended in 50 mM sodium acetate (pH 5.0).

[0164] Microscopy - Interactions in droplets and bulk solution. One sample was created, containing lignin, T. versicolor laccase (1 pM), and LB2-80 (0.5 mM), and split in two. LB2-80 was mixed in a ratio of 1:5 AF-488 labeled LB2-80 to unlabeled LB2-80. From this sample, we performed two experiments. One sample was emulsified in mineral oil containing 3 % (v / v) Abil EM 90 surfactant to produce stable aqueous microdroplets suitable for microscopy. The other sample was left as a bulk solution. LLPS was induced by incubating the emulsions at 35 °C for 30 min to allow formation of coacervate within the aqueous phase. Samples were imaged on an Olympus 1X83 epifluorescence microscope using the corresponding filter sets for AF488 and Cy3.Discussion.

[0165] FIGS. 11 A-B illustrate fluorescence microscopy images illustrating the interactions between lignin, Cy 3 -labeled T. versicolor laccase, and AF488-labeled LB2-80 in (A) microdroplets and (B) bulk solution. Both microdroplet and bulk solution samples were imaged at 35 °C for 30 minutes, depicting a colocalization of laccase and LB2-80 around lignin particles.

[0166] When LB2-80, T. versicolor laccase, and lignin were combined at low temperature, all components remained evenly distributed throughout the aqueous phase, indicating that the ELP was fully soluble and not interacting appreciably with lignin. When the temperature was raised to 35 °C to create coacervating conditions, the system underwent a clear structural transition from a uniform dispersion to one containing condensed polymer domains at the interfaces of lignin particles.

[0167] Upon heating above the transition temperature, LB2-80 condensed into micron-scale droplets that spontaneously reorganized around lignin particles. In the microdroplets, coacervate formation was readily observed as bright, punctate regions within the aqueous droplets. Fluorescence from AF488-labeled LB2-80 became concentrated at lignin particle boundaries, forming a continuous interfacial layer that visibly altered particle morphology. The colocalization of Cy3-labeled laccase with the LB2-80-rich regions confirmed that the enzy me partitioned into or was retained by the condensed phase. Microscopic observation revealed distinct changes in morphology consistent with interfacial enrichment and enzyme recruitment, confirming that coacervate formation localized catalytic components at lignin surfaces.

[0168] Importantly, similar behavior was observed in bulk solution, demonstrating that microdroplet confinement was not required to drive CatnC formation. In unconfined mixtures, LB2-80 underwent LLPS and formed visible condensed domains that adhered to lignin particles, producing comparable enrichment of both polymer and enzyme at the interface. This finding indicates that CatnCs can assemble spontaneously through intrinsic phase behavior and molecular affinity, rather than relying on external compartmentalization to enforce proximity.

[0169] Together, these results establish a mechanistic foundation for CatnC formation: upon phase separation. LB2-80 accumulates at lignin interfaces and simultaneously recruits soluble laccase into the same condensed phase. The resulting hybrid structures position enzymes in close proximity with the substrate surface, forming localized, enzy me-rich microenvironments capable of supporting efficient oxidative catalysis even in the absence of imposed physical confinement.Example VI-B, CatnCs: Polymer coacervates that coat lignin and incorporate enzy me hostages.

[0170] This Examples tests the hypothesis that SLAC 1.LB2-80 can bind to lignin particles and coat them upon LLPS, forming integrated CatnCs.

[0171] Materials and reagents. AF488-SLAC1.LB2-80: Alexa Fluor 488 fluorescently labeled SLAC1.LB2-80. SLAC1: a small laccase, prepared as stock solutions of 11 pM in IX PBS. Kraft lignin (Sigma- Aldrich): suspended in IX PBS.

[0172] Microscopy of microdroplets. Suspensions of kraft lignin were created by dispersing it in IX PBS and subjecting the suspension to multiple filtering steps to obtain suspensions with particles with sizes below 10 microns. The lignin suspension was mixed with either SLAC1.LB2-80 to a concentration of 0.25 mM or SLAC 1 to a concentration of 5.5 pM. SLAC1 LB2-80 was mixed in aratio of 1:5 AF-488 labeled SLAC1.LB2-80 to unlabeled SLAC1.LB2-80. The same mixing protocol was followed for SLAC1. The sample was emulsified in mineral oil containing 3% (v / v) Abil EM 90 surfactant to produce stable aqueous microdroplets suitable for microscopy. Preliminary images were taken before the temperature was increased to 45 °C, which is above the phase transition temperature. Additional images were taken periodically as LLPS occurred, monitoring the progression of coacervation. Samples were imaged on an Olympus 1X83 epifluorescence microscope using the corresponding filter sets for AF488 and Cy3.Discussion.

[0173] FIGS. 12A-B illustrate fluorescence microscopy images illustrating temperature-dependent LLPS of AF488-labeled SLAC1.LB2-80 anda lack of spatial association with kraft lignin particles, which autofluoresce in the Cy3 channel (FIG. 12A). AF488-labled SLAC1 was used as a control (FIG. 12B). Protein-lignin aggregates were noticed at high temperatures in the SLAC1.LB2-80 samples, but no such aggregates were seen in the SLAC1 control.

[0174] Microscopy analysis revealed that SLAC1.LB2-80 undergoes liquid-liquid phase separation under the tested conditions and forms phase separated assemblies that closely associate with dispersed lignin particles.Partial colocalization between the fluorescent signal of SLAC1.LB2-80 (AF488 channel) and the intrinsic autofluorescence of lignin (Cy3 channel) was observed, indicating that a fraction of the labeled protein-polymer associates with lignin surfaces.

[0175] However, a significant portion of the green fluorescence remained dispersed in the surrounding solution. It is hypothesized that this distribution arises because SLAC1.LB2-80 does not behave as atypical ELP forming fully reversible, liquid-like coacervates. Instead, only a subset of the polymer chains appears to interact and undergo phase separation, producing irregular, partially condensed structures, while a substantial fraction remains in the dilute phase. An additional possibility is that incomplete removal of free fluorophore during labeling contributed to residual background fluorescence in solution. The brightfield images provide more definitive evidence, showing clear phase-separated structures organized around lignin particles, supporting the formation of heterogeneous enzyme-polymer-lignin assemblies.

[0176] Unlike the previously characterized LB2-80 coacervates, which form reversible, spherical, liquid-like droplets, the SLAC1.LB2-80 assemblies exhibited irregular and partially fused morphologies with limited coalescence. The material had reduced mobility and loss of full reversibility upon cooling. This transition suggests that incorporation of the structured SLAC 1 enzyme domain into the intrinsically disordered ELP disrupts the dynamic phase behavior typical of purely disordered systems. Such solidification phenomena are commonly observed when intrinsically disordered polymers (IDPs) are fused to large, folded domains, as the ordered regions constrain molecular rearrangements and promote viscoelastic or arrested states.

[0177] Overall, these results demonstrate that SLAC1.LB2-80 selfassembles into enzyme-rich aggregates that organize around lignin particles, forming heterogeneous, surface-bound structures rather than fully coalesced, liquid-like coacervates. These assemblies retain selective lignin association and local enzyme enrichment but display reduced fluidity' and limited reversibility- compared to LB2-80 alone. The resulting lignin-associated SLAC1.LB2-80 complexes represent a new class of integrated catalytic materials (CatnCs) that covalently couple enzymatic activity, polymer phase behavior, and substrate affinityExample VI-C. CatnCs: Polymer coacervates that coat lignin and incorporate enzymes and redox mediators.

[0178] This Example tests the hypothesis that LB2-80 coacervates will simultaneously encapsulate lignin, laccase, and the redox mediator ABTS during phase separation, forming multicomponent CatnCs.

[0179] Materials and reagents. Kraft lignin (Sigma- Aldrich): resuspended in 50 mM sodium acetate buffer (pH 5.0). AF488-LB2-80: Alexa Fluor 488 fluorescently labeled LB2-80. Laccase from Trametes versicolor (>10 U mg '): prepared as a 10 pM stock solution in 50 mM sodium acetate buffer (pH 5.0). ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): prepared as a 10 mM stock solution in 50 mM sodium acetate buffer (pH 5.0).

[0180] Microfluidic microscopy. Suspensions of kraft lignin were created by dispersing it in sodium acetate and subjecting the suspension to multiple filtering steps to obtain suspensions with particles with sizes below 10 microns. T. versicolor laccase (1 pM), ABTS (0.5 mM), and LB2-80 (0.5 mM) were added immediately before droplet generation in a microfluidic droplet generator prior to loading of droplets into a microfluidic droplet parking chip. LB2-80 was mixed in a ratio of 1:5 AF-488 labeled LB2-80 to unlabeled LB2-80. The sample was emulsified in mineral oil containing 3 % (v / v) Abil EM 90 surfactant to produce stable aqueous droplets suitable for microscopy.Preliminary images were taken before the temperature was increased to 37 °C, which is above the phase transition temperature. Additional images w ere taken periodically as LLPS occurred, monitoring the progression of coacervation and the formation of the CatnC. Samples were imaged on an Olympus 1X83epifluorescence microscope using the corresponding filter sets for AF488 and Cy3.Discussion.

[0181] FIG. 13 illustrates fluorescence microscopy images showing the formation of CatnCs with autofluorescent lignin (Cy3 channel), AF488-labeled LB2-80, T. versicolor laccase, and ABTS. The images show clear encapsulation of the lignin particle within the coacervate and the change in the shape of the lignin particle.

[0182] Introducing both T. versicolor laccase and the redox mediator, ABTS, into the lignin / LB2-80 system produced multi-component coacervates that integrate substrate, enzyme, and mediator within a single condensed compartment. Before phase separation, the mixture remained optically homogeneous, with all species dispersed in the continuous aqueous phase.

[0183] Under coacervating conditions at 37 °C, LB2-80 condensed and reorganized around lignin particles to form well-defined compartments. Within these condensed regions, AF488-LB2-80 fluorescence concentrated at lignin interfaces, and lignin autofluorescence in the Cy3 channel extended into the condensed layer, consistent with encapsulation of lignin within the LB2-80-rich phase; incorporation of laccase and ABTS into this phase was established by independent partitioning assays rather than by direct fluorescence in these micrographs. The transformation of lignin particles from irregular aggregates to compacted, encapsulated structures further confirmed their incorporation into the dense phase.

[0184] This reorganization represents the spontaneous self-assembly of complete CatnCs, the protein-enzyme-mediator complexes that colocalize the key participants of oxidative catalysis. By bringing the laccase, its redox mediator, and the lignin substrate into a shared microenvironment, the coacervate establishes a kinetically favorable arrangement that minimizes diffusional barriers and supports sequential electron-transfer reactions. The resulting composite droplets therefore function as biomimetic catalytic compartments, where phase behavior of the polymer scaffold governs the spatial integration of enzymatic and redox processes essential for lignin oxidation.Example VII. CatnCs incorporating LB-ELP / Laccase / (ABTS) degrade lignin.Example VII-A, CatnCs incorporating LB-ELP / Laccase / ABTS bleach lignin.

[0185] This Example tests the hypothesis that oxidation of lignin in LB2-80 based CatnCs results in efficient bleaching by laccase and ABTS-mediated reactions.

[0186] Materials and reagents. Kraft lignin (Sigma- Aldrich): resuspended in 100 mM sodium acetate buffer (pH 5.0) to 40 mg ml.1. AF488-LB2-80: Alexa Fluor 488 fluorescently labeled LB2-80. Laccase from Trametes versicolor (>10 U mg '): prepared as a 10 pM stock solution in 100 mM sodium acetate buffer (pH 5.0). ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): prepared as a 1 mM stock solution in 100 mM sodium acetate buffer (pH 5.0).

[0187] Microscopy bleaching assay. Reactions were performed in 1.5 mL microcentrifuge tubes with a total volume of 62 pL. The following compositions were prepared: lignin + laccase (0.32 pM), lignin + laccase + ABTS (0.32 mM), lignin + laccase + LB2-80 (0.32 mM), and lignin + laccase + ABTS + LB2-80. All reactions were incubated at room temperature. Because ABTS depresses the temperature for LLPS of LB2-80 to below room temperature, this temperature is sufficient for forming CatnCs. At each time point (0 hr and 24 hr), a sample volume of 20 uL was extracted from the reaction mixture and emulsied in mineral oil containing 3 % (v / v) Abil EM 90 surfactant to produce stable aqueous microdroplets suitable for microscopy. Samples were imaged on an Olympus 1X83 epifluorescence microscope at room temperature using the corresponding filter sets for Cy3.

[0188] Lignin bleaching in microdroplets. Reactions were performed in 1.5 mL microcentrifuge tubes with a total volume of 31 pL. The following compositions were prepared: lignin + laccase (0.32 pM), lignin + laccase + ABTS (0.16 mM), lignin + laccase + LB2-80 (0.32 mM), and lignin + laccase + ABTS + LB2-80. LB2-80 was mixed in a ratio of 1:5 AF-488 labeled LB2-80 to unlabeled LB2-80. All reactions were made at room temperature. 5 pL of each sample was emulsified in mineral oil containing 3 % (v / v) Abil EM 90 surfactant to produce stable aqueous microdroplets suitable for microscopy. Samples wereimaged on an Olympus 1X83 epifluorescence microscope at 45 °C for 1 hour using the corresponding fdter sets for Cy3 and AF488.Discussion.

[0189] FIG. 14 illustrates fluorescence microscopy images of aqueous microdroplets in oil indicate time-dependent bleaching of lignin (which autofluoresces in the microscope’s Cy3 channel) in the presence of laccase, ABTS, and LB2-80 over 24 hours. A pronounced reduction in fluorescence intensity was observed across all conditions, consistent with progressive oxidation or degradation of lignin. In samples containing laccase, ABTS, and LB2-80, bleaching was most pronounced, and very’ few lignin particles were evident after 24 hours.

[0190] Oxidative bleaching of lignin was monitored over 24 hours to assess the catalytic activity of CatnCs containing LB2-80, T. versicolor laccase, and the mediator ABTS. In all samples containing laccase, a gradual decrease in lignin autofluorescence was observed, indicating progressive oxidation and structural modification of the aromatic substrate. However, the rate and extent of this bleaching varied across reaction conditions, reflecting the influence of both the mediator and LB2-80.

[0191] Reactions containing laccase and ABTS displayed similar fluorescence loss than those with laccase alone. Reactions containing laccase and LB2-80 supported similar bleaching efficiency, suggesting that coacervate formation marginally improved substrate-enzyme contact and may have stabilized active enzyme. The combination of all three components - laccase, ABTS, and LB2-80 - produced the most pronounced decrease in lignin signal after 24 hours, confirming that CatnCs promote localized oxidation through spatial colocalization of catalytic elements. Importantly, in these droplets, only- very few lignin particles were evident after 24 hours.

[0192] These results demonstrate that the LB2-80 coacervate scaffold acts as more than just a passive matrix: it enhances catalytic efficiency by colocalizing laccase, its mediator, and lignin within the same microenvironment. The visible bleaching and disappearance of lignin in the complete CatnC formulation provides functional evidence of cooperative enzymatic oxidation within the assembled compartmentalized system.

[0193] FIGS. 15A-B illustrate fluorescence imaging showing the loss of lignin fluorescence over a shorter timeframe (1 hour) in (A) a CatnC sample containing LB2-80, T. versicolor laccase, and ABTS, compared to (B) a control lacking LB2-80. A pronounced decrease in fluorescence is observed in the CatnC sample, while the control shows minimal change, suggesting lignin degradation in the presence of LB2-80.

[0194] To visualize lignin oxidation, coacervate-containing microdroplets were imaged during a 1-hour reaction at 45 °C. At the start of the assay, lignin particles exhibited strong autofluorescence within the Cy3 detection channel, and the surrounding medium appeared optically uniform. Over the course of incubation, the fluorescence intensity’ of lignin decreased markedly in droplets containing both LB2-80 and the laccase- ABTS pair, indicating active enzymatic oxidation within the coacervate environment.

[0195] Control samples lacking LB2-80 displayed minimal bleaching, even in the presence of laccase and ABTS, suggesting that oxidation efficiency is strongly dependent on the compartmentalized organization of the reaction components. In contrast, samples containing LB2-80 exhibited rapid loss of lignin fluorescence, confirming that the coacervate facilitates proximity -driven catalysis. The green AF488-LB2-80 signal remained concentrated at the lignin interface throughout the reaction, consistent with enzyme and mediator retention within the coacervate shell .

[0196] Taken together, these observations confirm that the complete LB2-80 / laccase / ABTS CatnC functions as an active oxidative microreactor. The polymer phase provides a dynamic yet stable matrix that maintains colocalization of catalytic species, supporting sustained electron transfer and substrate conversion. The observed fluorescence decay reflects real-time enzy matic bleaching of lignin within coacervate compartments, validating the functional integration of substrate, enzyme, and mediator achieved through CatnC assembly.Example VII-B, Visualization of product formation during catnc-mediated lignin oxidation.

[0197] This Examples tests the hypothesis that fluorescence detectable with the DAPI filter set will appear only after coacervation and enzy maticoxidation, indicating the formation and accumulation of oxidation products within LB2-80-based CatnCs.

[0198] Materials and reagents. Kraft lignin (Sigma- Aldrich): resuspended in 50 mM Sodium Acetate buffer (pH 5.0). AF488-LB2-80: Alexa Fluor 488 fluorescently labeled LB2-80. Laccase from Trametes versicolor (>10 U mg '): prepared as a 10 pM stock solution in 50 mM sodium acetate buffer (pH 5.0). ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): prepared as a 2 mM stock solution in 50 mM sodium acetate buffer (pH 5.0).

[0199] Microflui die microscopy. Suspensions of kraft lignin were created by dispersing it in sodium acetate and subjecting the suspension to multiple filtering steps to obtain suspensions with particles with sizes below 10 microns. T versicolor laccase (1 pM), ABTS (0.5 mM), and LB2-80 (0.5 mM) were added immediately before droplet generation and loading of droplets onto a microfluidic parking chip. LB2-80 was mixed in a ratio of 1:5 AF-488 labeled LB2-80 to unlabeled LB2-80. The sample was emulsified in mineral oil containing 3% (v / v) Abil EM 90 surfactant to produce stable aqueous droplets suitable for microscopy. Preliminary images were taken before the temperature was increased to 37 °C, which is above the phase transition temperature.Additional images were taken periodically as LLPS occurred, monitoring the progression of coacervation and the formation of the CatnC. Samples w ere imaged on an Olympus 1X83 epifluorescence microscope using the corresponding filter sets for DAPI, AF488 and Cy3.Discussion.

[0200] FIG. 16 illustrates fluorescence microscopy images depicting the appearance of product fluorescence during lignin oxidation within LB2-80-based CatnCs containing Cy3-autofluorescent lignin and AF488-labeled LB2-80. Samples were imaged at 10 °C and 37 °C using DAPI. Cy3, and AF488 filter sets. Fluorescence detected in the DAPI channel emerged only after coacervation at 37 °C, becoming prominent after 30 minutes, which coincides with lignin and LB2-80 localization, suggesting the formation of new oxidized or conjugated lignin products.

[0201] At 10 °C, below the transition temperature of LB2-80, samples containing lignin, ABTS, LB2-80, and laccase appeared optically uniform withno evidence of phase separation in the solution. Fluorescence imaging showed a diffuse AF488 signal from solvated LB2-80 throughout the aqueous phase and faint, particle-localized emission from lignin in the Cy3 channel consistent with lignin autofluorescence. Importantly, little signal was detected using the DAPI filter set at this stage, establishing that the starting mixture lacks blue-emitting species and that any subsequent DAPI-channel signal reflects newly formed autofluorescent products.

[0202] When samples were heated to 37 °C, LB2-80 underwent LLPS to form coacervate domains that reorganized around lignin particles. Concomitant with coacervate formation, the AF488 signal intensified at particle boundaries, indicating interfacial accumulation of LB2-80 and the emergence of a continuous coating. In parallel, two reproducible spectral / contrast changes were observed: (i) particle-associated emission redistributed and diminished in negative contrast as lignin became enveloped by the condensed phase, and (ii) a previously absent DAPI-channel signal appeared and localized to regions containing both lignin and condensed LB2-80. The onset of blue-channel autofluorescence only after LLPS indicates that the condensed, enzyme-enriched microenvironment supports chemical transformations of lignin that yield products with excitation / emission characteristics detectable by the DAPI filter set.

[0203] Taken together, the absence of DAPI-channel fluorescence in the initial mixed state, and its appearance only after coacervation at 37 °C, suggests that CatnC assembly is directly coupled to product formation and accumulation. These observations provide evidence that LB2-80 coacervates function as oxidative microreactors.Example VII-C. Colorimetric and visual studies of lignin degradation in LB2-80 / laccase-based CatnCs.

[0204] This Example evaluates the effect of the LB2-80 on laccase-mediated lignin degradation under conditions above the transition temperature (Tt) of LB2-80, where the protein forms a coacervate phase. The goal was to document that the presence and concentration of LB2-80 modulate enzymatic activity, lignin oxidation, or the solubilization of degradation products.

[0205] Materials and reagents. Kraft lignin (Sigma-Aldrich): resuspended in 50mM sodium acetate. Laccase from Trametes versicolor (>10 prepared as a 20 pM stock solution in 50 mM sodium acetate buffer (pH 5.0). LB2-80: an elastin-like polypeptide containing a lignin-binding domain, dissolved in IX PBS and added to final concentrations of 0.5 mM.

[0206] Experimental design. Reactions were performed in 1.5 mL microcentrifuge tubes with a total volume of 200 pL. The following compositions were prepared in triplicate for each condition: lignin + laccase (2 pM), and lignin + laccase + LB2-80 (0.5 mM). All reactions were incubated at room temperature, above the transition temperature (Tt) of LB2-80, to induce coacervation, for 10 hours. Initial measurements (time Oh) were collected immediately after mixing and analyzed alongside the 10-hour timepoints.

[0207] Analytical methods. Lignin degradation and enzymatic oxidation were monitored by UV-Vis spectroscopy using an Agilent Synergy7Hl microplate reader. Extinction was monitored at 340 nm. a wavelength sensitive to the presence and formation of aromatic conjugated structures, phenolic intermediates, and lignin-derived aldehydes. Changes at this wavelength report on both oxidative breakdown and structural modification of lignin polymers. Degradation was expressed as a relative change in extinction between the 0-hour and 10-hour samples. At the end of the 10-hour incubation, photographs were taken to visually assess lignin changes in the reaction mixtures. Images w ere taken of the tubes after 10 hours to compare samples with and without the laccase system and to observe visible changes in lignin, dispersion, or solubility.Discussion.

[0208] FIG. 17A illustrates quantitative analysis of lignin degradation in the presence of LB2-80 and laccase, after 10 h incubation at 26 °C (above the phase separation temperature (Tt)). as measured by extinction at 340 nm. FIG.17B illustrates photographs showing macroscopic appearance of laccase-lignin reactions in the presence of LB2-80 after 10 h incubation above the phase separation temperature (Tt) in microcentrifuge tubes. FIG. 17C illustrates photographs show ing macroscopic appearance of laccase-lignin samples in the presence of LB2-80 after 10 h incubation above the phase separation temperature (Tt) in microtiter plate wells. FIG. 17D illustrates photographs showingmacroscopic appearance of control, laccase-free samples including lignin and LB2-80 after 10 h incubation above the phase separation temperature (Tt) in microtiter plate wells

[0209] In reactions containing lignin and laccase, spectral measurements at 340 nm revealed a consistent decrease in extinction after 10 hours in the presence of LB2-80, whereas control samples lacking the polymer showed a slight increase (FIG. 17A). The 340 nm region reflects the presence of conjugated aromatic and phenolic structures in lignin as well as oxidation-derived intermediates such as aldehydes and quinonoid species. Therefore, a decrease at this wavelength indicates oxidative conversion or breakdown of these aromatic species. In contrast, the small extinction increase observed without LB2-80 suggests accumulation of soluble conjugated intermediates, consistent with incomplete oxidation. The extent of these spectral changes scaled with LB2-80 concentration: higher concentrations produced greater decreases in 340 nm extinction, implying enhanced oxidative turnover.

[0210] Visual inspection of the reaction mixtures corroborated the spectroscopic data (FIGS. 17B-D). For examples containing lignin and laccase, the mixtures with LB2-80 appeared lighter in color and less aggregated after incubation, whereas those without LB2-80 remained darker and more turbid. This visible clarification corresponds to enhanced oxidative processing and dispersal of lignin particles within the coacervate phase, suggesting that LB2-80 enhances enzyme efficiency. The spectroscopic and visual evidence demonstrate that the presence of LB2-80 above its transition temperature markedly influences the extent of lignin degradation. By concentrating reactive components within the coacervate phase, LB2-80 enables more complete oxidation of lignin, producing a visibly clearer and chemically more oxidized product mixture.Example VII-D. Detecting carbonyl-containing lignin degradation products by 2,4-dinitrophenylhydrazine (DNPH) assay.

[0211] This Example tests the hypothesis that aldehyde and ketone products generated during laccase- and ABTS-mediated oxidation of lignin can be quantified by DNPH derivatization to assess both the extent of oxidation andthe distribution of products between the LB2-80 coacervate and supernatant phases.

[0212] Materials and reagents. Kraft lignin (Sigma- Aldrich): resuspended in 50 mM sodium acetate (pH 5.0) to 10 mg mL1and diluted to a final concentration of 5 mg mL1during sample prep. Laccase from Trametes versicolor (>10 U mg1): prepared as a 200 pM stock solution in 50 mM sodium acetate buffer (pH 5.0) and diluted to a final concentration of 10 pM during sample prep. ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): prepared as a 2 mM stock solution in 50 mM sodium acetate and diluted to 0.5 mM during sample prep. LB2-80: an elastin-like polypeptide containing a lignin-binding domain, dissolved in PBS at a concentration of 2 mM and diluted to a final concentration of 0.5 mM during sample prep. 2,4-Dinitrophenol (2,4-DNP): a chemical compound used as a reagent to test for the presence of aldehydes and ketones.

[0213] Experimental procedure. After incubating reactions at 45 °C for 1 h (to form the coacervate), phases were separated, the coacervate was resuspended to the original reaction volume, and the following were analyzed: lignin in sodium acetate (control), lignin + laccase + ABTS, lignin + laccase + ABTS + LB2-80 (coacervate fraction, resuspended), and lignin + laccase + ABTS + LB2-80 (supernatant fraction). To perform the DNPH assay. 20 pL of each sample was added to 30 pL of 100 mM HC1. 50 pL of 1 mM 2,4-DNP was added to each tube and incubated for 5 minutes at room temperature. After incubation, 100 pL of IM NaOH was added to balance the pH. Extinction was measured at 450 nm using an Agilent Synergy Hl microplate reader. All samples were measured in triplicate.Discussion.

[0214] FIG. 18 illustrates extinction measurements at 450 nm illustrating the intensity for a lignin control, bulk solution reaction control, and between the coacervate (pellet) and supernatant fractions for LB2-80. The resuspended LB2-80 pellet showed the highest signal, suggesting the greatest production of aldehyde / ketone products.

[0215] For the lignin control, minimal color change and low extinction at 450 nm were observed, consistent with the limited carbonyl content ofunmodified kraft lignin. In contrast, samples containing lignin, laccase, and ABTS exhibited a clear increase in extinction intensity, indicating the oxidative conversion of lignin into aldehyde- and ketone-containing intermediates. This trend agrees with the expected laccase-catalyzed oxidation of phenolic moieties and subsequent cleavage or modification of aromatic rings.

[0216] When LB2-80 was included and the reaction mixture was phase-separated into coacervate (pellet) and supernatant fractions, a pronounced difference emerged. The coacervate fraction displayed the highest extinction at 450 nm, whereas the supernatant fraction exhibited substantially lower extinction. Because the DNPH assay detects hydrazone adducts formed with carbonyl groups, this result indicates that the majority of aldehyde- and ketone-bearing products were retained within the coacervate phase rather than remaining in bulk solution. The elevated signal in the pellet fraction suggests that oxidation and subsequent carbonyl formation are most concentrated within the LB2-80 condensate microenvironment, where the enzyme and mediator are co-localized with lignin.

[0217] This distribution pattern highlights the functional role of the coacervate as a reactive compartment that both enhances oxidative turnover and transiently retains the resulting soluble or partially oxidized lignin fragments. In contrast, the lower extinction in the supernatant implies that comparatively few carbonyl-containing products escaped into the surrounding solution, reinforcing that catalytic activity and product accumulation are spatially confined within the CatnC.

[0218] Overall, the DNPH analysis demonstrates that greater oxidative conversion of lignin occurs within the LB2-80 coacervate phase compared to reactions lacking the polymer scaffold. These findings support the concept that phase-separated CatnCs act as localized oxidative microreactors, concentrating substrates, enzymes and mediators to promote carbonyl product generation and retention within the condensed phase.

[0219] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modificationsare possible within the scope of the embodiments of the present invention. Thus, it should be understood that although the present invention has been specifically disclosed by specific embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary' skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present invention.Exemplary Embodiments.

[0220] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0221] Aspect 1 provide a method of breaking down at least one component of a substrate, the method comprising:providing a catabolic nanocompartment comprising a liquid coacervate phase, at least one catalyst within the coacervate phase, and an aqueous phase surrounding the coacervate phase; andcontacting the catabolic nanocompartment and the substrate together such that the catalyst breaks down the at least one component of the substrate and forms degradation products of the at least one component.

[0222] Aspect 2 provides the method of Aspect 1, wherein the substrate comprises a biomass product, a synthetic material to be decomposed or recycled, an environmental contaminant, atoxic contaminant, a biological molecule, a pathological biological entity7(e.g., pathogens or cancer cells), or a combination thereof.

[0223] Aspect 3 provides the method of any one of Aspects 1-2. wherein the substrate comprises woody biomass.

[0224] Aspect 4 provides the method of any one of Aspects 1-3, wherein the substrate comprises solid lignin, solubilized lignin, solid kraft lignin, solubilized kraft lignin, extracted lignin, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfomc acid), wood, wood particles, woody biomass, or a combination thereof.

[0225] Aspect 5 provides the method of any one of Aspects 1-4, wherein the substrate comprises woody biomass, the at least one component comprises lignin, and wherein the degradation products of the at least one component comprise lignin degradation products.

[0226] Aspect 6 provides the method of any one of Aspects 1-5, wherein the coacervate phase further comprises one or more polymers.

[0227] Aspect 7 provides the method of Aspect 6, wherein the one or more polymers comprise one or more water-soluble polymers.

[0228] Aspect 8 provides the method of any one of Aspects 6-7, wherein the one or more polymers comprise a synthetic polymer, a recombinantly synthesized biopolymer, or a combination thereof.

[0229] Aspect 9 provides the method of any one of Aspects 6-8, wherein the one or more polymers comprise a mussel foot protein, a resilin-like peptide, an elastin-like peptide, poly(A-isopropyl acry lamide), polylysine, polyglutamic acid, poly(2-dimethylamino)ethyl methacrylate, gum Arabic, polyethyleneimine, polystyrene sulfonate, poly(ethylene glycol), polylacrylic acid), a lignin-binding elastin-like polypeptide (LB-ELP), a cellulose-binding elastin-like polypeptide (CB-ELP), a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP), a small laccase / elastin-like polypeptide (SLAC-ELP). or a combination thereof.

[0230] Aspect 10 provides the method of any one of Aspects 6-9, wherein the one or more polymers comprise a mussel foot protein, a resilin-like peptide, an elastin-like peptide, poly(A-isopropyl acrylamide), or a combination thereof.

[0231] Aspect 11 provides the method of any one of Aspects 6-10, wherein the one or more polymers comprise one or more pairs of polymers chosen from polylysine / polyglutamic acid, poly(2-dimethylamino)ethyl methacrylate / gum arabic. poly ethyleneimine / poly styrene sulfonate, and poly(ethylene glycol) / poly(acrylic acid).

[0232] Aspect 12 provides the method of any one of Aspects 6-11, wherein the one or more polymers comprise one or more lignin-binding elastinlike polypeptides (LB-ELP) chosen from LB 1-40, LB 1-60, LB 1-80, LB2-40, LB2-60, LB2-80, or a combination thereof.

[0233] Aspect 13 provides the method of any one of Aspects 6-12, wherein the one or more polymers comprise a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP) or a small laccase / elastin-like polypeptide (SLAC-ELP), wherein the SLAC-LB-ELP or SLAC-ELP comprises SLAC1.LB1-40, SLAC1.LB1-60, SLAC1.LB1-80, SLAC1.LB2-40,SLAC1.LB2-60, SLAC1.LB2-80, SLAC.E1-40, SLAC.E1-80, or a combination thereof.

[0234] Aspect 14 provides the method of any one of Aspects 1-13, wherein the catalyst comprises an enzy me, a non-enzymatic catalyst (e.g., a metal-organic compound), or a combination thereof.

[0235] Aspect 15 provides the method of any one of Aspects 1-14, wherein the catalyst comprises an enzyme.

[0236] Aspect 16 provides the method of any one of Aspects 1-15, wherein the catalyst comprises an enzy me comprising a laccase, a small laccase (SLAC), a peroxidase, or a combination thereof.

[0237] Aspect 17 provides the method of any one of Aspects 1-16, wherein the catalyst is covalently attached to one or more polymers in the coacervate phase.

[0238] Aspect 18 provides the method of 17, wherein the catalyst comprises a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP), a small laccase / elastin-like polypeptide (SLAC-ELP), or a combination thereof.

[0239] Aspect 19 provides the method of any one of Aspects 1-18, wherein the catalyst is partitioned into the coacervate phase through favorable noncovalent interactions with the coacervate phase and free of covalent attachment to one or more polymers in the coacervate phase.

[0240] Aspect 20 provides the method of any one of Aspects 1-19, wherein the coacervate phase further comprises a redox mediator.

[0241] Aspect 21 provides the method of Aspect 20, wherein the redox mediator facilitates electron transfer between the catalyst and the substrate, enabling oxidation of high-molecular- weight or sterically inaccessible domains of the substrate.

[0242] Aspect 22 provides the method of any one of Aspects 20-21, wherein the redox mediator comprises 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldehyde (SGA), 1 -hydroxy benzotriazole (HBT), 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), 2,6-dimethoxyphenol (DMP), or a combination thereof.

[0243] Aspect 23 provides the method of any one of Aspects 1-22, wherein repolymerization of the degradation products of the at least onecomponent is decreased and / or prevented compared to exposure of the substrate to the at least one catalyst outside of the catabolic nanocompartment.

[0244] Aspect 24 provides the method of any one of Aspects 1-23, wherein the aqueous phase comprises a deep eutectic solvent.

[0245] Aspect 25 provides the method of Aspect 24, wherein the deep eutectic solvent comprises:a hydrogen bond donor and a hydrogen bond acceptor.

[0246] Aspect 26 provides the method of Aspect 25, wherein the hydrogen bond donor is chosen from ethylene glycol, glycerol, erythritol, xylitol, and propionic acid.

[0247] Aspect 27 provides the method of any one of Aspects 25-26. wherein the hydrogen bond acceptor is chosen from choline chloride, choline dihydrogen citrate, and a betaine.

[0248] Aspect 28 provides the method of any one of Aspects 1-27, wherein the coacervate phase comprises a cellulose binding domain.

[0249] Aspect 29 provides the method of any one of Aspects 1-28, wherein the coacervate phase comprises an oxygen reservoir.

[0250] Aspect 30 provides the method of Aspect 29, wherein the oxygen reservoir comprises silicone nanoparticles.

[0251] Aspect 31 provides the method of any one of Aspects 1-30, wherein the coacervate phase comprises a spreading promoter.

[0252] Aspect 32 provides the method of any one of Aspects 1-31, further comprising milling a substrate precursor into particles to form the substrate.

[0253] Aspect 33 provides the method of any one of Aspects 1-32, further comprising collecting the degradation products of the at least one component.

[0254] Aspect 34 provides a method of degrading lignin in woody biomass comprising:providing a catabolic nanocompartment comprising a liquid coacervate phase, at least one laccase enzyme within the coacervate phase, and an aqueous phase surrounding the coacervate phase;contacting the catabolic nanocompartment and a woody biomass particle comprising lignin together such that the laccase enzyme degrades the lignin and forms lignin degradation products.

[0255] Aspect 35 provides the method of Aspect 34, wherein repolymerization of the lignin degradation products is decreased and / or prevented compared to exposure of the woody biomass particle to the one or more laccase enzymes outside of the catabolic nanocompartment.

[0256] Aspect 36 provides the method of any one of Aspects 34-35, wherein the laccase enzyme comprises a small laccase (SLAC).

[0257] Aspect 37 provides the method of any one of Aspects 34-36, wherein the laccase enzyme comprises a fusion protein comprising:a laccase domain; anda coacervation tag domain.

[0258] Aspect 38 provides the method of any one of Aspects 34-37, wherein the coacervate phase comprises at least one component chosen from polylysine, polyglutamic acid, mussel foot protein, resilin-like peptides, elastinlike peptides, poly(2-dimethylamino)ethyl methacrylate, gum arabic, polyethyleneimine, polysty rene sulfonate, poly(ethylene glycol), poly(acrylic acid), and poly(N-isopropyl acrylamide).

[0259] Aspect 39 provides the method of any one of Aspects 34-38. wherein the liquid coacervate phase comprises at least one redox mediator.

[0260] Aspect 40 provides the method of Aspect 39, wherein the redox mediator is chosen from 1 -hydroxy benzotriazole (HBT) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).

[0261] Aspect 41 provides the method of any one of Aspects 34-40, further comprisingmilling woody materials into particles to form the woody biomass particle.

[0262] Aspect 42 provides the method of any one of Aspects 34-41, further comprising collecting the lignin degradation products.

[0263] Aspect 43 provide a catabolic nanocompartment composition comprising:a liquid coacervate phase comprising at least one catalyst; and an aqueous phase surrounding the coacervate phase;wherein the composition degrades at least one component of a substrate to form degradation products of the at least one component when the at least one component is contacted with the liquid coacervate phase.

[0264] Aspect 44 provides a catabolic nanocompartment composition comprising:a liquid coacervate phase comprising at least one laccase enzyme; and an aqueous phase surrounding the coacervate phase;wherein the composition degrades lignin to form lignin degradation products when the lignin is contacted with the liquid coacervate phase.

[0265] Aspect 45 provides the catabolic nanocompartment composition of Aspect 44, wherein when the lignin is contacted with the liquid coacervate phase and the lignin degradation products are formed, repolymerization of the lignin degradation products is decreased and / or prevented compared to exposure of the woody biomass particle to the one or more laccase enzymes outside of the catabolic nanocompartment.

[0266] Aspect 46 provides a method of making a catabolic nanocompartment composition comprising:providing an aqueous solution comprising coacervate-forming components; andadding at least one catalyst to form a liquid coacervate phase containing the catalyst, to form the catabolic nanocompartment composition;wherein the catabolic nanocompartment composition degrades at least one component of a substrate to form degradation products of the at least one component when the at least one component is contacted with the liquid coacervate phase.

[0267] Aspect 47 provides a method of making a catabolic nanocompartment composition comprising:providing an aqueous solution comprising coacervate-forming components; andadding at least one laccase enzyme to form a liquid coacervate phase containing the laccase enzyme, to form the catabolic nanocompartment composition;wherein the catabolic nanocompartment composition degrades lignin to form lignin degradation products when the lignin is contacted with the liquid coacervate phase.

[0268] Aspect 48 provides the method or composition of any one or any combination of Aspects 1-47 optionally configured such that all elements or options recited are available to use or select from.

Claims

CLAIMSWhat is claimed is:

1. A method of breaking down at least one component of a substrate, the method comprising:providing a catabolic nanocompartment comprising a liquid coacervate phase, at least one catalyst within the coacervate phase, and an aqueous phase surrounding the coacervate phase; andcontacting the catabolic nanocompartment and the substrate together such that the catalyst breaks down the at least one component of the substrate and forms degradation products of the at least one component.

2. The method of claim 1, wherein the substrate comprises a biomass product, a synthetic material to be decomposed or recycled, an environmental contaminant, a toxic contaminant, a biological molecule, a pathological biological entity, or a combination thereof.

3. The method of claim 1, wherein the substrate comprises wood, wood particles, woody biomass, or a combination thereof, the at least one component comprises lignin, and wherein the degradation products of the at least one component comprise lignin degradation products.

4. The method of claim 1, wherein the coacervate phase further comprises one or more polymers.

5. The method of claim 4, wherein the one or more polymers comprise a mussel foot protein, a resilin-like peptide, an elastin-like peptide, poly(N-isopropyl acrylamide), polylysine, polyglutamic acid, poly(2-dimethylamino)ethyl methacrylate, gum Arabic, polyethyleneimine, polystyrene sulfonate, poly(ethylene glycol), poly(acrylic acid), a lignin-binding elastin-like polypeptide (LB-ELP), a cellulose-binding elastin-like polypeptide (CB-ELP), a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP), a small laccase / elastin-like polypeptide (SLAC-ELP), or a combination thereof.

6. The method of claim 4, wherein the one or more polymers comprise one or more lignin-binding elastin-like polypeptides (LB-ELP) chosen from LB 1-40, LB 1-60, LB 1-80, LB2-40, LB2-60, LB2-80, or a combination thereof.

7. The method of claim 4, wherein the one or more polymers comprise a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP) or a small laccase / elastin-like polypeptide (SLAC-ELP), wherein the SLAC-LB-ELP or SLAC-ELP comprises SLAC1.LB1-40, SLAC1.LB1-60, SLAC1.LB1-80, SLAC1.LB2-40, SLAC1.LB2-60, SLAC1.LB2-80, SLAC.E1-40, SLAC. El-80, or a combination thereof.

8. The method of claim 1, wherein the catalyst comprises an enzyme.

9. The method of claim 8, wherein the enzyme comprises a laccase, a small laccase (SLAC), a peroxidase, or a combination thereof.

10. The method of claim 1, wherein the catalyst is covalently attached to one or more polymers in the coacervate phase.

11. The method of claim 1, wherein the catalyst comprises a small laccase / lignin-binding elastin-like polypeptide (SLAC-LB-ELP), a small laccase / elastin-like polypeptide (SLAC-ELP), or a combination thereof.

12. The method of claim 1, wherein the catalyst is partitioned into the coacervate phase through favorable noncovalent interactions with the coacervate phase and free of covalent attachment to one or more polymers in the coacervate phase.

13. The method of claim 1, wherein the coacervate phase further comprises a redox mediator.

14. The method of claim 1, wherein repolymerization of the degradation products of the at least one component is decreased and / or prevented compared to exposure of the substrate to the at least one catalyst outside of the catabolic nanocompartment.

15. The method of claim 1, wherein the aqueous phase comprises a deep eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor is chosen from ethylene glycol, glycerol, erythritol, xylitol, and propionic acid, and wherein the hydrogen bond acceptor is chosen from choline chloride, choline dihydrogen citrate, and a betaine.

16. The method of claim 1, further comprising milling a substrate precursor into particles to form the substrate.

17. The method of claim 1, further comprising collecting the degradation products of the at least one component.

18. The method of claim 1 , whereinthe substrate comprises wood, wood particles, woody biomass, or a combination thereof,the at least one component comprises lignin,the degradation products of the at least one component comprise lignin degradation products, andthe at least one catalyst comprises a laccase enzyme.

19. A catabolic nanocompartment composition comprising:a liquid coacervate phase comprising at least one catalyst; and an aqueous phase surrounding the coacervate phase;wherein the composition degrades at least one component of a substrate to form degradation products of the at least one component when the at least one component is contacted with the liquid coacervate phase.

20. A method of making a catabolic nanocompartment composition comprising:providing an aqueous solution comprising coacervate-forming components; andadding at least one catalyst to form a liquid coacervate phase containing the catalyst, to form the catabolic nanocompartment composition;wherein the catabolic nanocompartment composition degrades at least one component of a substrate to form degradation products of the at least one component when the at least one component is contacted with the liquid coacervate phase.

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